Precipitable peptides

ABSTRACT

The invention is directed to a Ca2+ precipitable polypeptide tags and cassettes useful for purification of molecules from heterogeneous samples. The invention also relates to methods for bioseparation of molecules comprising Ca2+ precipitable tags and cassettes.

This application is a continuation-in-part of International ApplicationNo. PCT/US2012/033293, filed Apr. 12, 2012, which claims priority toU.S. provisional application Ser. No. 61/475,042 filed Apr. 13, 2011,and also claims the benefit of and priority to U.S. provisionalapplication Ser. No. 61/616,341 filed Mar. 27, 2012, the disclosures ofall of which are hereby incorporated by reference in their entiretiesfor all purposes.

This invention was made with government support under grant numberW9132T-08-2-0012 awarded by the DTRA and under grant numberW9132T-08-2-0002 awarded by the US Army. The government has certainrights in the invention.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art as known to those skilled therein as of the date of theinvention described herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is incorporated byreference in its entirety. Said ASCII copy, created on Jan. 16, 2014, isnamed 19240.924US3_SL.txt and is 335,744 bytes in size.

BACKGROUND OF THE INVENTION

Rapid protein purification is an important requirement in manybioengineering applications where significant amounts of time arecurrently spent purifying proteins from heterogeneous samples. There arecurrently a number of approaches for performing bioseparation, but theseapproaches are expensive, time consuming, can require specializedtreatments.

A variety of approaches currently exist for purifying recombinantproteins such as using a poly-histidine tag, glutathione S-transferase(GST) fusions or fusion to an elastin-like peptide (ELP). In the case ofELPs, a fusion protein can be precipitated from solution by increasingthe temperature of the sample (Banki, et al., Nat Meth, vol. 2, no. 9,pp. 659-662, 2005; Fong et al, Trends in Biotechnology, vol. 28, no. 5,pp. 272-279, May 2010). One limitation of ELP technology is thatincreased temperature can adversely affect the stability of fusionproteins. Another limitation of ELP technology is that inducingtemperature changes are difficult in large scale preparations.

There is a need for improved purification methods for rapid purificationof molecules (e.g. exogenously expressed proteins) from heterogeneoussamples in a rapid manner and with high levels of recovery. Thisinvention addresses these needs.

SUMMARY OF THE INVENTION

In certain aspects, the invention relates to a precipitable beta rollcassette (PBRC) comprising one or more beta roll tags (PBRTs) whereinthe one or more PBRTs comprise the amino acid sequence of SEQ ID NO: 1

In certain aspects, the invention relates to a precipitable beta rollcassette (PBRC) comprising one or more beta roll tags (PBRTs) whereinthe one or more PBRTs are independently any of: (a) a polypeptide havingthe amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having theamino acid sequence of any of SEQ ID NOs: 25-1337

In certain aspects, the invention relates to a precipitable beta rollcassette (PBRC) comprising one or more beta roll tags (PBRTs) whereinthe one or more PBRTs are independently any of: (a) a polypeptide havingthe amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having theamino acid sequence of any of SEQ ID NOs 25-1337, (c) a polypeptidecomprising the amino acid sequence G (SEQ ID NO: 1343), wherein, (i) theX at position 2 is an amino acid selected from the group consisting ofglycine, asparagine or aspartic acid, and (ii) the X at position 3 is anamino acid selected from the group consisting of alanine, serine,glycine, aspartic acid, glutamic acid, leucine or asparagine, and (iii)the X at position 4 is an amino acid selected from the group consistingof glycine or alanine, and (iv) the X at position 5 is an amino acidselected from the group consisting of asparagine, aspartic acid,alanine, or serine, and (v) the X at position 6 is an amino acidselected from the group consisting of aspartic acid or asparagine, (vi)the X at position 7 is an amino acid selected from the group consistingof threonine, isoleucine, valine, or leucine, and (vii) the X atposition 8 is an amino acid selected from the group consisting ofleucine, isoleucine, or phenylalanine, and (viii) the X at position 9 isan amino acid selected from the group consisting of tyrosine,isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid,lysine or serine.

In certain aspects, the invention relates to a precipitable beta rollcassette (PBRC) comprising one or more beta roll tags (PBRTs) whereinthe one or more PBRTs are independently any of: (a) a polypeptide havingthe amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having theamino acid sequence of any of SEQ ID NOs 25-1337, (c) a polypeptidecomprising the amino acid sequence G (SEQ ID NO: 1343), wherein, (i) theX at position 2 is an amino acid selected from the group consisting ofglycine, asparagine or aspartic acid, and (ii) the X at position 3 is anamino acid selected from the group consisting of alanine, serine,glycine, aspartic acid, glutamic acid, leucine or asparagine, and (iii)the X at position 4 is an amino acid selected from the group consistingof glycine or alanine, and (iv) the X at position 5 is an amino acidselected from the group consisting of asparagine, aspartic acid,alanine, or serine, and (v) the X at position 6 is an amino acidselected from the group consisting of aspartic acid or asparagine, (vi)the X at position 7 is an amino acid selected from the group consistingof threonine, isoleucine, valine, or leucine, and (vii) the X atposition 8 is an amino acid selected from the group consisting ofleucine, isoleucine, or phenylalanine, and (viii) the X at position 9 isan amino acid selected from the group consisting of tyrosine,isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid,lysine or serine, or (c) a variant PBRT.

In certain aspects, the invention relates to a precipitable beta rollcassette (PBRC) comprising one or more beta roll tags (PBRTs) whereinthe one or more PBRTs are independently any of: (a) a polypeptide havingthe amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having theamino acid sequence of any of SEQ ID NOs 25-1337, (c) a polypeptidecomprising the amino acid sequence G (SEQ ID NO: 1343), wherein, (i) theX at position 2 is an amino acid selected from the group consisting ofglycine, asparagine or aspartic acid, and (ii) the X at position 3 is anamino acid selected from the group consisting of alanine, serine,glycine, aspartic acid, glutamic acid, leucine or asparagine, and (iii)the X at position 4 is an amino acid selected from the group consistingof glycine or alanine, and (iv) the X at position 5 is an amino acidselected from the group consisting of asparagine, aspartic acid,alanine, or serine, and (v) the X at position 6 is an amino acidselected from the group consisting of aspartic acid or asparagine, (vi)the X at position 7 is an amino acid selected from the group consistingof threonine, isoleucine, valine, or leucine, and (vii) the X atposition 8 is an amino acid selected from the group consisting ofleucine, isoleucine, or phenylalanine, and (viii) the X at position 9 isan amino acid selected from the group consisting of tyrosine,isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid,lysine or serine, or (c) a variant PBRT, wherein the PBRC furthercomprises a capping sequence.

In certain aspects, the invention relates to a precipitable beta rollcassette (PBRC) comprising one or more beta roll tags (PBRTs) whereinthe one or more PBRTs are independently any of: (a) a polypeptide havingthe amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having theamino acid sequence of any of SEQ ID NOs 25-1337, (c) a polypeptidecomprising the amino acid sequence G (SEQ ID NO: 1343), wherein, (i) theX at position 2 is an amino acid selected from the group consisting ofglycine, asparagine or aspartic acid, and (ii) the X at position 3 is anamino acid selected from the group consisting of alanine, serine,glycine, aspartic acid, glutamic acid, leucine or asparagine, and (iii)the X at position 4 is an amino acid selected from the group consistingof glycine or alanine, and (iv) the X at position 5 is an amino acidselected from the group consisting of asparagine, aspartic acid,alanine, or serine, and (v) the X at position 6 is an amino acidselected from the group consisting of aspartic acid or asparagine, (vi)the X at position 7 is an amino acid selected from the group consistingof threonine, isoleucine, valine, or leucine, and (vii) the X atposition 8 is an amino acid selected from the group consisting ofleucine, isoleucine, or phenylalanine, and (viii) the X at position 9 isan amino acid selected from the group consisting of tyrosine,isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid,lysine or serine, or (c) a variant PBRT, wherein the PBRC furthercomprises a stabilizing polypeptide.

In certain aspects, the invention relates to a PBRC linked purificationmoiety comprising a precipitable beta roll cassette (PBRC) comprisingone or more beta roll tags (PBRTs) wherein the one or more PBRTs areindependently any of: (a) a polypeptide having the amino acid sequenceof SEQ ID NO: 1, or (b) a polypeptide having the amino acid sequence ofany of SEQ ID NOs 25-1337, (c) a polypeptide comprising the amino acidsequence G (SEQ ID NO: 1343), wherein, (i) the X at position 2 is anamino acid selected from the group consisting of glycine, asparagine oraspartic acid, and (ii) the X at position 3 is an amino acid selectedfrom the group consisting of alanine, serine, glycine, aspartic acid,glutamic acid, leucine or asparagine, and (iii) the X at position 4 isan amino acid selected from the group consisting of glycine or alanine,and (iv) the X at position 5 is an amino acid selected from the groupconsisting of asparagine, aspartic acid, alanine, or serine, and (v) theX at position 6 is an amino acid selected from the group consisting ofaspartic acid or asparagine, (vi) the X at position 7 is an amino acidselected from the group consisting of threonine, isoleucine, valine, orleucine, and (vii) the X at position 8 is an amino acid selected fromthe group consisting of leucine, isoleucine, or phenylalanine, and(viii) the X at position 9 is an amino acid selected from the groupconsisting of tyrosine, isoleucine, valine, phenylalanine, threonine,asparagine, aspartic acid, lysine or serine, or (c) a variant PBRT. Incertain embodiments, the PBRC is linked to the purification moiety by apeptide bond. In certain embodiments, the PBRC is linked to thepurification moiety by a chemical bond that is not a peptide bond.

In certain aspects, the invention relates to a PBRC linked purificationmoiety comprising a precipitable beta roll cassette (PBRC) comprisingone or more beta roll tags (PBRTs) wherein the one or more PBRTs areindependently any of: (a) a polypeptide having the amino acid sequenceof SEQ ID NO: 1, or (b) a polypeptide having the amino acid sequence ofany of SEQ ID NOs 25-1337, (c) a polypeptide comprising the amino acidsequence G (SEQ ID NO: 1343), wherein, (i) the X at position 2 is anamino acid selected from the group consisting of glycine, asparagine oraspartic acid, and (ii) the X at position 3 is an amino acid selectedfrom the group consisting of alanine, serine, glycine, aspartic acid,glutamic acid, leucine or asparagine, and (iii) the X at position 4 isan amino acid selected from the group consisting of glycine or alanine,and (iv) the X at position 5 is an amino acid selected from the groupconsisting of asparagine, aspartic acid, alanine, or serine, and (v) theX at position 6 is an amino acid selected from the group consisting ofaspartic acid or asparagine, (vi) the X at position 7 is an amino acidselected from the group consisting of threonine, isoleucine, valine, orleucine, and (vii) the X at position 8 is an amino acid selected fromthe group consisting of leucine, isoleucine, or phenylalanine, and(viii) the X at position 9 is an amino acid selected from the groupconsisting of tyrosine, isoleucine, valine, phenylalanine, threonine,asparagine, aspartic acid, lysine or serine, or (c) a variant PBRT,wherein the PBRC further comprises a cleavage site located N-terminallyor C-terminally to one or more of the one or more PBRTs. In certainembodiments, the cleavage site is selected from the group comprising anintein cleavage site, a Factor Xa cleavage site, a thrombin cleavagesite, an enterokinase cleavage site, or a signal peptidase cleavagesite.

In certain aspects, the invention relates to a polypeptide comprising aPBRC linked purification moiety comprising a precipitable beta rollcassette (PBRC) comprising one or more beta roll tags (PBRTs) whereinthe one or more PBRTs are independently any of: (a) a polypeptide havingthe amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having theamino acid sequence of any of SEQ ID NOs 25-1337, (c) a polypeptidecomprising the amino acid sequence G (SEQ ID NO: 1343), wherein, (i) theX at position 2 is an amino acid selected from the group consisting ofglycine, asparagine or aspartic acid, and (ii) the X at position 3 is anamino acid selected from the group consisting of alanine, serine,glycine, aspartic acid, glutamic acid, leucine or asparagine, and (iii)the X at position 4 is an amino acid selected from the group consistingof glycine or alanine, and (iv) the X at position 5 is an amino acidselected from the group consisting of asparagine, aspartic acid,alanine, or serine, and (v) the X at position 6 is an amino acidselected from the group consisting of aspartic acid or asparagine, (vi)the X at position 7 is an amino acid selected from the group consistingof threonine, isoleucine, valine, or leucine, and (vii) the X atposition 8 is an amino acid selected from the group consisting ofleucine, isoleucine, or phenylalanine, and (viii) the X at position 9 isan amino acid selected from the group consisting of tyrosine,isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid,lysine or serine, or (c) a variant PBRT; and a purification moiety.

In certain aspects, the invention relates to a nucleic acid encoding anyof the polypeptides described herein.

In certain embodiments, the invention relates to a method for purifyinga PBRC linked purification moiety, the method comprising (a) expressingthe PBRC linked purification moiety in an expression system, (b)collecting the PBRC linked purification moiety in a first medium, (c)adding Ca2+ to the first medium so as to induce precipitation of PBRClinked purification moiety, (d) removing unprecipiated material from themedium from the precipitated PBRC linked purification moiety, (e)resuspending the PBRC linked purification moiety in a second mediumhaving a lower than the free Ca2+ concentration than the free Ca2+concentration obtained after step (c). In certain embodiments, a calciumchelator is added to the second medium of step (e). In certainembodiments, steps (c) to (e) are repeated one or more times. In certainembodiments, the method further comprises a step of removingprecipitated material between step (b) and step (c). In certainembodiments, the PBRC comprises a cleavage site between the PBRC and thepurification moiety. In certain embodiments, the method furthercomprises steps of: (i) cleaving the PBRC linked purification moiety soas to separate the purification moiety from the PBRC, (ii) adding Ca2+to the medium so as to induce precipitation of the PBRC, and (iii)isolating the unprecipiated purification moiety. In certain embodiments,the cleavage site is an intein cleavage site.

In certain aspects, the invention relates to an expression vectorcomprising, as arranged from 5′ to 3′, a promoter, a nucleic acidsequence encoding the PBRC of any of claims 1-4, and at least onecloning site.

In certain aspects, the invention relates to an expression vectorcomprising, as arranged from 5′ to 3′, a promoter, at least one cloningsite, and a nucleic acid sequence encoding the PBRC of any of claims1-4.

In certain aspects, the invention relates to an expression vectorcomprising, as arranged from 5′ to 3′, a promoter, at least one cloningsite, a nucleic acid sequence encoding the PBRC of any of claims 1-4 andat least a second cloning site.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B show a SDS-PAGE gel showing purification of a moleculecomprising a precipitable beta-roll tag. FIG. 1A shows purification ofprecipitatable maltose binding protein comprising a precipitatable betaroll tag and an enterokinase cleavage site (MBP-PBRT). Total lysate isshown in lane 1 and lanes 2-7 are precipitation/wash cycles. After twocycles, the sample consists nearly only of the MBP fusion protein withthe precipitating tag attached (MBP-PBRT). Recovery is nearly 100% ofthe expressed protein. FIG. 1B shows SDS-PAGE analysis of purifiedMBP-PBRT and MBP-PBRT subjected to digestion of the enterokinasedigestion site (FIG. 1B). Lane 1 shows purified MBP-PBRT, lane 2 showssupernatant after overnight digest and lane 3 shows the pellet.

FIG. 2 shows a SDS-PAGE gel showing a successful purification of apolypeptide comprising a 5 or 17 repeat C-capped precipitable beta-rolltags. Lanes (from left to right): 1. MBP-5cap lysate, 2. MBP-5capsupernatant, 3. MBP-5C resuspended precipitate. The 4-6 lanes are thesame expect with the capped 17 repeat construct.

FIG. 3 is circular dichroism data (CD) showing precipitation of apolypeptide comprising a 17 repeat C-capped precipitable beta-roll tag(GGAGNDTLY)17INAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGIEKLVEAMAQYPD (SEQ ID NO: 1344) out of solution withincreasing calcium concentrations from 0 to 100 mM calcium. Loss ofspectra indicates that the peptide is precipitating out of solution andno longer visible via CD.

FIG. 4 shows a GGXGXDXXX (SEQ ID NO: 2) sequence heat map. The heat mapwas determined by using BLAST to find beta roll sequences similar to themetalloprotease of S. marcescens and then quantifying the frequency ofamino acids at each of the nine positions after beta roll sequences wereidentified. FIG. 4 discloses the consensus sequence as SEQ IN NO: 1343.

FIG. 5 shows a schematic illustration of the corkscrew configuration oftandem Ca2+ binding sequences. The figure shows a crystal structureimage of the beta roll domain from the metalloprotease of S. marcescens(1SAT in the Protein Databank). Alternating 9-amino acid repeats arehighlighted in green and red. Coordinated calcium ions are in white. Theimage represents 5 repeats of beta roll sequence. While there is nocrystal structure for the adenylate cyclase beta roll domain, the highdegree of sequence similarity to the consensus beta roll indicates thatthe adenylate cyclase beta roll domain is similar.

FIG. 6 shows a schematic illustration of the corkscrew configuration oftandem Ca2+ binding sequences from a different angle than shown in FIG.5. The 6^(th) residue binds the calcium ion. The 7th and 9th residues ofeach repeat are those that face outwards. The 8^(th) residue is buriedin the hydrophobic core. These residues are threonine and tyrosine,respectively in SEQ ID NO: 1.

FIG. 7 shows the full crystal structure of the metalloprotease of S.marcescens. The black spheres indicate the position of the calcium ionswithin the beta roll domain.

FIGS. 8A-8D show a characterization of beta roll distribution, sequencedeviation and number of repeats. FIG. 8A shows a distribution of beatroll lengths as frequency plotted against the number of beta rollrepeats. FIG. 8B shows beta roll sequence deviation from consensus byposition plotted as proportion deviation as a function of distance fromterminus. FIG. 8C shows a probability of deviation from consensusplotted as probability as a function of the number of beta roll repeats.FIG. 8D shows amount of beta roll in overall protein plotted as thenumber of beta roll residues as a function of the number of totalresidues.

FIG. 9 shows an exemplary protocol for purification of a polypeptidecomprising a precipitable beta roll tag. The images depict precipitationof maltose binding protein fused to seventeen repeats of a PBRT. ThePBRC comprises 17 repeats of the amino acid sequence of SEQ ID NO: 1.The PBRC does not comprise a capping sequence.

FIG. 10 shows purification of a maltose binding protein/PBRT/greenfluorescent protein fusion (MBP-PBRT-GFP). FIG. 10A shows aprecipitation and resuspension of the MBP-PBRT-GFP polypeptide. FIG. 10Bshows SDS-PAGE of multiple precipitation wash cycles. The lanes of theSDS gel are as follows: lane 1—ladder; lane 2—clarified lysate; lane3—precipitation in 25 mM calcium followed by three washes; lane4—precipitation in 50 mM calcium followed by three washes; lane5—precipitation in 75 mM calcium followed by three washes; lane—6precipitation in 100 mM calcium followed by three washes. All lanes arenormalized in terms of concentration. Recovery percentage can beestimated by comparing the band intensity in lane 2 to subsequent lanes.

FIG. 11 shows non-limiting examples of polypeptides comprising a PBRTsuitable for use with the methods described herein. FIG. 11 disclosesSEQ ID NO: 1368.

FIGS. 12A-12G show vector Maps at shown in FIG. 12A-G. Vector maps aregiven for each construct prepared, highlighting the important features.

FIG. 13A-13B show beta roll structure and sequence logo. FIG. 13A showscrystal structure of β roll domain from metalloprotease of S. marcescens(PDB: 1 SAT). FIG. 13B shows amino acid frequencies for single β rollrepeat identifying consensus sequence GGAGNDTLY (SEQ ID NO: 1). Heightof the letter corresponds to proportion of sequences containing theparticular amino acid at that position.

FIG. 14 shows the role of PBRT length in precipitation. Mass ofprecipitated pellet vs. calcium chloride concentration and PBRT size.Results for MBP-PBRT5(1), MBP-PBRT9(●), MBP-PBRT13(∘), andMBP-PBRT17(▾). Error bars represent standard deviations for 3 trials.

FIG. 15 shows ion specificity of PBRT precipitation. PurifiedMBP-PBRT17-GFP was mixed with 100 mM of the compound indicated, andcentrifuged to collect any pellet. The tube was then inverted so thatprecipitated protein remained on top. The compounds were as follows: (a)CaCl2, (b) MgCl2, (c) MnCl2, (d) NaCl, and (e) (NH4)2SO4

FIG. 16 shows SDS-PAGE results for purification of the four constructstested. Numbers are standard size in kDa. Expected molecular weights forMBP-PBRT17, MBP-PBRT17-GFP, MBP-PBRT17-βlac, and MBP-PBRT17-AdhD are57.1, 83.4, 88.6, and 89.1 kDa, respectively. (1-2) Purification ofMBP-PBRT 17. Lane 1 is clarified lysate, and Lane 2 is purified fusionprotein. (3-4) Same samples for purification of MBP-PBRT17-GFP. (5-6)Same samples for MBP-PBRT17-βlac. (7-8) Same samples forMBP-PBRT17-AdhD.

FIG. 17 shows SDS-PAGE results for purification and cleavage of AdhD.Numbers are standard size in kDa. Estimated molecular weight for AdhD is31.9 kDa. (1) Clarified MBP-PBRT17-AdhD lysate. (2) Purified fusionprotein. (3) Enterokinase cleavage. (4) Precipitated MBP-PBRT17. (5)Soluble AdhD. 3× protein concentrations were used in lanes 3, 4, and 5.

DETAILED DESCRIPTION OF THE INVENTION

The issued patents, applications, and other publications that are citedherein are hereby incorporated by reference to the same extent as ifeach was specifically and individually indicated to be incorporated byreference.

Purification is a major requirement in many bioengineering applicationswhere significant amounts of time are currently spent purifying proteinsfrom heterogeneous samples. The invention described herein relates tomethods for rapidly purifying a purification moiety (e.g. a targetpolypeptide) from a heterogeneous medium using a PBRC. For example, incertain embodiments, a target polypeptide can be produced as a fusionprotein in frame with a PBRC. In certain embodiments, the fusion proteincomprising the target protein and the PBRC can further comprise aspecific cleavage site (e.g. an intein cleavage site or an enterokinasecleavage site) between the target protein sequence and the PBRCsequence. In such embodiments, cleavage at the cleavage site can be usedto separate the PBRC from the target polypeptide.

The beta-roll domain is a right-handed beta helix found in a number ofproteins. The consensus sequence for beta-roll peptides is tandemrepeats of the 9 amino acid sequence GGXGXDX(L/F/I)X (SEQ ID NO: 24). Inthe presence of calcium, the conformation aligns to adopt the helicalturns. Two repeats of the sequence are required to make a completehelical turn and each of these turns binds a calcium atom. In theabsence of calcium, the peptide exists in a disordered conformation.Therefore the β-roll domain exhibits natural allosteric regulation. Asynthetic version of the β-roll peptide has been produced with 8 repeatsof GGSGNDNLS (SEQ ID NO: 1338) and this peptide was found to bindcalcium and fold into the β-roll structure (Lilie et al., FEBS Lett 470(2), 173 (2000)). The domain is capable of reversibly unfolding uponremoval of the calcium. Beta roll sequences are known to play a role insecretion as part of the bacterial Type I secretion system (Davidson, etal., Microbiol. Mol. Biol. Rev. 72 (2008), pp. 317-364; Holland et al.,Mol. Membr. Biol. 22 (2005), pp. 29-39; Chenal, et al., J. Biol. Chem.284(2009), pp. 1781-1789; Welch, Pore-Forming Toxins 257 (2001), pp.85-111; Rose et al., J. Biol. Chem. 270 (1995), pp. 26370-26376;Baumann, J. Mol. Biol. 242 (1994), pp. 244-251; Angkawidjaja, et al.,FEBS Lett. 581(2007), pp. 5060-5064; Meier et al., J. Biol. Chem. 282(2007), pp. 31477-31483; Bauche et al., J. Biol. Chem. 281 (2006), pp.16914-16926; Baumann et al., EMBO J. 12 (1993), pp. 3357-3364;Angkawidjaja et al., FEBS Lett. 579(2005), pp. 4707-4712).

The precipitable-beta roll tags and precipitable-beta roll cassettesdescribed herein are class of designed peptides which possess theability to reversibly precipitate in response to calcium ions. In oneaspect, the invention described herein relates to the surprising findingthat PBRCs (e.g. PBRTs repeats of sequence GGAGNDTLY (SEQ ID NO: 1))undergo reversible precipitation upon calcium binding. In anotheraspect, the invention described herein relates to the surprising findingthat attachment of a PBRC to a second molecule (e.g. attachment to aprotein as a fusion protein comprising an in-frame) can be used topurify the second molecule through reversible precipitation. In anotheraspect, the invention described herein relates to the use of calciumconcentration changes at room temperature to induce precipitation ofrecombinant molecules comprising a precipitable beta-roll tag.

In addition to target polypeptides, the PBRCs describe herein are alsosuitable for purifying non-peptide purification moieties of widelyvarying types, including, for example, lipids, oligonucleotides andcarbohydrates, small organic or inorganic molecules, proteins,single-stranded or double-stranded oligonucleotides, polynucleotides. Incertain aspects, applications for the methods and compositions describedherein include, but are not limited to, the purification of recombinantproteins the removal of target proteins from a sample, and detection ofcompounds for diagnostic purposes. The invention also extends to theantibodies that specifically bind to a PBRT or a PBRC and the methodsfor using the PBRTs and PBRCs described herein.

Without wishing to be bound to theory, in certain embodiments, the PBRTsand PBRCs described herein can undergo a reversible Ca2+ bindingdependent transition wherein they are structurally disordered and highlysoluble in a medium below a Ca2+ concentration (or free Ca2+) transitionconcentration, but exhibit a disorder to order phase transition when theCa2+ or free Ca2+ concentration is raised above the Ca2+ (or free Ca2+)transition concentration. Again, without wishing to be bound by theory,in some embodiments, the disorder to order phase transition leads toprecipitation of the PBRTs or PBRCs. Precipitation of PBRC can be usedto remove and isolated them from solution (e.g. by centrifugation). Inone embodiment, the invention described herein relates to a PBRC whichfunctions reversible Ca2+ precipitable tag when linked to a purificationmoiety of interest. In embodiments where the PBRC is linked to apurification moiety of interest, the methods described herein can beused to induce precipitation of the PBRC linked purification moiety.Because the transition concentration dependent phase transition isreversible, the PBRT and PBRC can be resolubilized in a medium having aCa2+ concentration (or free Ca2+) below the transition concentration. Incertain embodiments, this can be accomplished by introducing mediumhaving reduced, or no Ca2+, or by removing, or chelating Ca2+ from themedium. When the precipitate is resuspended in calcium-free buffer or ina buffer comprising a calcium ion chelator (e.g. EGTA or EDTA), theprecipitate resuspends into solution.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20%.

In certain embodiments, the term “precipitable-beta roll tag” (PBRT)refers to an amino acid sequence having the sequence GGAGNDTLY (SEQ IDNO: 1). In certain embodiments, a PBRT refers to an amino acid sequencehaving the amino acid sequence GXXXXXXXX (SEQ ID NO: 1343), wherein (a)the X at position 2 is an amino acid selected from the group consistingof glycine, asparagine or aspartic acid, and (b) the X at position 3 isan amino acid selected from the group consisting of alanine, serine,glycine, aspartic acid, glutamic acid, leucine or asparagine, and (c)the X at position 4 is an amino acid selected from the group consistingof glycine or alanine, and (d) the X at position 5 is an amino acidselected from the group consisting of asparagine, aspartic acid,alanine, or serine, and (e) the X at position 6 is an amino acidselected from the group consisting of aspartic acid or asparagine, (f)the X at position 7 is an amino acid selected from the group consistingof threonine, isoleucine, valine, or leucine, and (g) the X at position8 is an amino acid selected from the group consisting of leucine,isoleucine, or phenylalanine, and (h) the X at position 9 is an aminoacid selected from the group consisting of tyrosine, isoleucine, valine,phenylalanine, threonine, asparagine, aspartic acid, lysine or serine,or a nucleic acid encoding the same. A PBRT refers to an amino acidsequence having the sequence set forth in any of SEQ ID NOs: 25-1337.

TABLE 1 SEQ ID NOs: 25-1337 Sequence (SEQ ID NO) GDNASDLFS SEQ ID NO: 25GNAGNDTLY SEQ ID NO: 26 GDAGNDTLY SEQ ID NO: 27 GGSGNDTLY SEQ ID NO: 28GNSGNDTLY SEQ ID NO: 29 GDSGNDTLY SEQ ID NO: 30 GGGGNDTLY SEQ ID NO: 31GNGGNDTLY SEQ ID NO: 32 GDGGNDTLY SEQ ID NO: 33 GGDGNDTLY SEQ ID NO: 34GNDGNDTLY SEQ ID NO: 35 GDDGNDTLY SEQ ID NO: 36 GGAGDDTLY SEQ ID NO: 37GNAGDDTLY SEQ ID NO: 38 GDAGDDTLY SEQ ID NO: 39 GGSGDDTLY SEQ ID NO: 40GNSGDDTLY SEQ ID NO: 41 GDSGDDTLY SEQ ID NO: 42 GGGGDDTLY SEQ ID NO: 43GNGGDDTLY SEQ ID NO: 44 GDGGDDTLY SEQ ID NO: 45 GGDGDDTLY SEQ ID NO: 46GNDGDDTLY SEQ ID NO: 47 GDDGDDTLY SEQ ID NO: 48 GGAGADTLY SEQ ID NO: 49GNAGADTLY SEQ ID NO: 50 GDAGADTLY SEQ ID NO: 51 GGSGADTLY SEQ ID NO: 52GNSGADTLY SEQ ID NO: 53 GDSGADTLY SEQ ID NO: 54 GGGGADTLY SEQ ID NO: 55GNGGADTLY SEQ ID NO: 56 GDGGADTLY SEQ ID NO: 57 GGDGADTLY SEQ ID NO: 58GNDGADTLY SEQ ID NO: 59 GDDGADTLY SEQ ID NO: 60 GGAGNNTLY SEQ ID NO: 61GNAGNNTLY SEQ ID NO: 62 GDAGNNTLY SEQ ID NO: 63 GGSGNNTLY SEQ ID NO: 64GNSGNNTLY SEQ ID NO: 65 GDSGNNTLY SEQ ID NO: 66 GGGGNNTLY SEQ ID NO: 67GNGGNNTLY SEQ ID NO: 68 GDGGNNTLY SEQ ID NO: 69 GGDGNNTLY SEQ ID NO: 70GNDGNNTLY SEQ ID NO: 71 GDDGNNTLY SEQ ID NO: 72 GGAGDNTLY SEQ ID NO: 73GNAGDNTLY SEQ ID NO: 74 GDAGDNTLY SEQ ID NO: 75 GGSGDNTLY SEQ ID NO: 76GNSGDNTLY SEQ ID NO: 77 GDSGDNTLY SEQ ID NO: 78 GGGGDNTLY SEQ ID NO: 79GNGGDNTLY SEQ ID NO: 80 GDGGDNTLY SEQ ID NO: 81 GGDGDNTLY SEQ ID NO: 82GNDGDNTLY SEQ ID NO: 83 GDDGDNTLY SEQ ID NO: 84 GGAGANTLY SEQ ID NO: 85GNAGANTLY SEQ ID NO: 86 GDAGANTLY SEQ ID NO: 87 GGSGANTLY SEQ ID NO: 88GNSGANTLY SEQ ID NO: 89 GDSGANTLY SEQ ID NO: 90 GGGGANTLY SEQ ID NO: 91GNGGANTLY SEQ ID NO: 92 GDGGANTLY SEQ ID NO: 93 GGDGANTLY SEQ ID NO: 94GNDGANTLY SEQ ID NO: 95 GDDGANTLY SEQ ID NO: 96 GGAGNDILY SEQ ID NO: 97GNAGNDILY SEQ ID NO: 98 GDAGNDILY SEQ ID NO: 99 GGSGNDILY SEQ ID NO: 100GNSGNDILY SEQ ID NO: 101 GDSGNDILY SEQ ID NO: 102GGGGNDILY SEQ ID NO: 103 GNGGNDILY SEQ ID NO: 104GDGGNDILY SEQ ID NO: 105 GGDGNDILY SEQ ID NO: 106GNDGNDILY SEQ ID NO: 107 GDDGNDILY SEQ ID NO: 108GGAGDDILY SEQ ID NO: 109 GNAGDDILY SEQ ID NO: 110GDAGDDILY SEQ ID NO: 111 GGSGDDILY SEQ ID NO: 112GNSGDDILY SEQ ID NO: 113 GDSGDDILY SEQ ID NO: 114GGGGDDILY SEQ ID NO: 115 GNGGDDILY SEQ ID NO: 116GDGGDDILY SEQ ID NO: 117 GGDGDDILY SEQ ID NO: 118GNDGDDILY SEQ ID NO: 119 GDDGDDILY SEQ ID NO: 120GGAGADILY SEQ ID NO: 121 GNAGADILY SEQ ID NO: 122GDAGADILY SEQ ID NO: 123 GGSGADILY SEQ ID NO: 124GNSGADILY SEQ ID NO: 125 GDSGADILY SEQ ID NO: 126GGGGADILY SEQ ID NO: 127 GNGGADILY SEQ ID NO: 128GDGGADILY SEQ ID NO: 129 GGDGADILY SEQ ID NO: 130GNDGADILY SEQ ID NO: 131 GDDGADILY SEQ ID NO: 132GGAGNNILY SEQ ID NO: 133 GNAGNNILY SEQ ID NO: 134GDAGNNILY SEQ ID NO: 135 GGSGNNILY SEQ ID NO: 136GNSGNNILY SEQ ID NO: 137 GDSGNNILY SEQ ID NO: 138GGGGNNILY SEQ ID NO: 139 GNGGNNILY SEQ ID NO: 140GDGGNNILY SEQ ID NO: 141 GGDGNNILY SEQ ID NO: 142GNDGNNILY SEQ ID NO: 143 GDDGNNILY SEQ ID NO: 144GGAGDNILY SEQ ID NO: 145 GNAGDNILY SEQ ID NO: 146GDAGDNILY SEQ ID NO: 147 GGSGDNILY SEQ ID NO: 148GNSGDNILY SEQ ID NO: 149 GDSGDNILY SEQ ID NO: 150GGGGDNILY SEQ ID NO: 151 GNGGDNILY SEQ ID NO: 152GDGGDNILY SEQ ID NO: 153 GGDGDNILY SEQ ID NO: 154GNDGDNILY SEQ ID NO: 155 GDDGDNILY SEQ ID NO: 156GGAGANILY SEQ ID NO: 157 GNAGANILY SEQ ID NO: 158GDAGANILY SEQ ID NO: 159 GGSGANILY SEQ ID NO: 160GNSGANILY SEQ ID NO: 161 GDSGANILY SEQ ID NO: 162GGGGANILY SEQ ID NO: 163 GNGGANILY SEQ ID NO: 164GDGGANILY SEQ ID NO: 165 GGDGANILY SEQ ID NO: 166GNDGANILY SEQ ID NO: 167 GDDGANILY SEQ ID NO: 168GGAGNDVLY SEQ ID NO: 169 GNAGNDVLY SEQ ID NO: 170GDAGNDVLY SEQ ID NO: 171 GGSGNDVLY SEQ ID NO: 172GNSGNDVLY SEQ ID NO: 173 GDSGNDVLY SEQ ID NO: 174GGGGNDVLY SEQ ID NO: 175 GNGGNDVLY SEQ ID NO: 176GDGGNDVLY SEQ ID NO: 177 GGDGNDVLY SEQ ID NO: 178GNDGNDVLY SEQ ID NO: 179 GDDGNDVLY SEQ ID NO: 180GGAGDDVLY SEQ ID NO: 181 GNAGDDVLY SEQ ID NO: 182GDAGDDVLY SEQ ID NO: 183 GGSGDDVLY SEQ ID NO: 184GNSGDDVLY SEQ ID NO: 185 GDSGDDVLY SEQ ID NO: 186GGGGDDVLY SEQ ID NO: 187 GNGGDDVLY SEQ ID NO: 188GDGGDDVLY SEQ ID NO: 189 GGDGDDVLY SEQ ID NO: 190GNDGDDVLY SEQ ID NO: 191 GDDGDDVLY SEQ ID NO: 192GGAGADVLY SEQ ID NO: 193 GNAGADVLY SEQ ID NO: 194GDAGADVLY SEQ ID NO: 195 GGSGADVLY SEQ ID NO: 196GNSGADVLY SEQ ID NO: 197 GDSGADVLY SEQ ID NO: 198GGGGADVLY SEQ ID NO: 199 GNGGADVLY SEQ ID NO: 200GDGGADVLY SEQ ID NO: 201 GGDGADVLY SEQ ID NO: 202GNDGADVLY SEQ ID NO: 203 GDDGADVLY SEQ ID NO: 204GGAGNNVLY SEQ ID NO: 205 GNAGNNVLY SEQ ID NO: 206GDAGNNVLY SEQ ID NO: 207 GGSGNNVLY SEQ ID NO: 208GNSGNNVLY SEQ ID NO: 209 GDSGNNVLY SEQ ID NO: 210GGGGNNVLY SEQ ID NO: 211 GNGGNNVLY SEQ ID NO: 212GDGGNNVLY SEQ ID NO: 213 GGDGNNVLY SEQ ID NO: 214GNDGNNVLY SEQ ID NO: 215 GDDGNNVLY SEQ ID NO: 216GGAGDNVLY SEQ ID NO: 217 GNAGDNVLY SEQ ID NO: 218GDAGDNVLY SEQ ID NO: 219 GGSGDNVLY SEQ ID NO: 220GNSGDNVLY SEQ ID NO: 221 GDSGDNVLY SEQ ID NO: 222GGGGDNVLY SEQ ID NO: 223 GNGGDNVLY SEQ ID NO: 224GDGGDNVLY SEQ ID NO: 225 GGDGDNVLY SEQ ID NO: 226GNDGDNVLY SEQ ID NO: 227 GDDGDNVLY SEQ ID NO: 228GGAGANVLY SEQ ID NO: 229 GNAGANVLY SEQ ID NO: 230GDAGANVLY SEQ ID NO: 231 GGSGANVLY SEQ ID NO: 232GNSGANVLY SEQ ID NO: 233 GDSGANVLY SEQ ID NO: 234GGGGANVLY SEQ ID NO: 235 GNGGANVLY SEQ ID NO: 236GDGGANVLY SEQ ID NO: 237 GGDGANVLY SEQ ID NO: 238GNDGANVLY SEQ ID NO: 239 GDDGANVLY SEQ ID NO: 240GGAGNDTIY SEQ ID NO: 241 GNAGNDTIY SEQ ID NO: 242GDAGNDTIY SEQ ID NO: 243 GGSGNDTIY SEQ ID NO: 244GNSGNDTIY SEQ ID NO: 245 GDSGNDTIY SEQ ID NO: 246GGGGNDTIY SEQ ID NO: 247 GNGGNDTIY SEQ ID NO: 248GDGGNDTIY SEQ ID NO: 249 GGDGNDTIY SEQ ID NO: 250GNDGNDTIY SEQ ID NO: 251 GDDGNDTIY SEQ ID NO: 252GGAGDDTIY SEQ ID NO: 253 GNAGDDTIY SEQ ID NO: 254GDAGDDTIY SEQ ID NO: 255 GGSGDDTIY SEQ ID NO: 256GNSGDDTIY SEQ ID NO: 257 GDSGDDTIY SEQ ID NO: 258GGGGDDTIY SEQ ID NO: 259 GNGGDDTIY SEQ ID NO: 260GDGGDDTIY SEQ ID NO: 261 GGDGDDTIY SEQ ID NO: 262GNDGDDTIY SEQ ID NO: 263 GDDGDDTIY SEQ ID NO: 264GGAGADTIY SEQ ID NO: 265 GNAGADTIY SEQ ID NO: 266GDAGADTIY SEQ ID NO: 267 GGSGADTIY SEQ ID NO: 268GNSGADTIY SEQ ID NO: 269 GDSGADTIY SEQ ID NO: 270GGGGADTIY SEQ ID NO: 271 GNGGADTIY SEQ ID NO: 272GDGGADTIY SEQ ID NO: 273 GGDGADTIY SEQ ID NO: 274GNDGADTIY SEQ ID NO: 275 GDDGADTIY SEQ ID NO: 276GGAGNNTIY SEQ ID NO: 277 GNAGNNTIY SEQ ID NO: 278GDAGNNTIY SEQ ID NO: 279 GGSGNNTIY SEQ ID NO: 280GNSGNNTIY SEQ ID NO: 281 GDSGNNTIY SEQ ID NO: 282GGGGNNTIY SEQ ID NO: 283 GNGGNNTIY SEQ ID NO: 284GDGGNNTIY SEQ ID NO: 285 GGDGNNTIY SEQ ID NO: 286GNDGNNTIY SEQ ID NO: 287 GDDGNNTIY SEQ ID NO: 288GGAGDNTIY SEQ ID NO: 289 GNAGDNTIY SEQ ID NO: 290GDAGDNTIY SEQ ID NO: 291 GGSGDNTIY SEQ ID NO: 292GNSGDNTIY SEQ ID NO: 293 GDSGDNTIY SEQ ID NO: 294GGGGDNTIY SEQ ID NO: 295 GNGGDNTIY SEQ ID NO: 296GDGGDNTIY SEQ ID NO: 297 GGDGDNTIY SEQ ID NO: 298GNDGDNTIY SEQ ID NO: 299 GDDGDNTIY SEQ ID NO: 300GGAGANTIY SEQ ID NO: 301 GNAGANTIY SEQ ID NO: 302GDAGANTIY SEQ ID NO: 303 GGSGANTIY SEQ ID NO: 304GNSGANTIY SEQ ID NO: 305 GDSGANTIY SEQ ID NO: 306GGGGANTIY SEQ ID NO: 307 GNGGANTIY SEQ ID NO: 308GDGGANTIY SEQ ID NO: 309 GGDGANTIY SEQ ID NO: 310GNDGANTIY SEQ ID NO: 311 GDDGANTIY SEQ ID NO: 312GGAGNDIIY SEQ ID NO: 313 GNAGNDIIY SEQ ID NO: 314GDAGNDIIY SEQ ID NO: 315 GGSGNDIIY SEQ ID NO: 316GNSGNDIIY SEQ ID NO: 317 GDSGNDIIY SEQ ID NO: 318GGGGNDIIY SEQ ID NO: 319 GNGGNDIIY SEQ ID NO: 320GDGGNDIIY SEQ ID NO: 321 GGDGNDIIY SEQ ID NO: 322GNDGNDIIY SEQ ID NO: 323 GDDGNDIIY SEQ ID NO: 324GGAGDDIIY SEQ ID NO: 325 GNAGDDIIY SEQ ID NO: 326GDAGDDIIY SEQ ID NO: 327 GGSGDDIIY SEQ ID NO: 328GNSGDDIIY SEQ ID NO: 329 GDSGDDIIY SEQ ID NO: 330GGGGDDIIY SEQ ID NO: 331 GNGGDDIIY SEQ ID NO: 332GDGGDDIIY SEQ ID NO: 333 GGDGDDIIY SEQ ID NO: 334GNDGDDIIY SEQ ID NO: 335 GDDGDDIIY SEQ ID NO: 336GGAGADIIY SEQ ID NO: 337 GNAGADIIY SEQ ID NO: 338GDAGADIIY SEQ ID NO: 339 GGSGADIIY SEQ ID NO: 340GNSGADIIY SEQ ID NO: 341 GDSGADIIY SEQ ID NO: 342GGGGADIIY SEQ ID NO: 343 GNGGADIIY SEQ ID NO: 344GDGGADIIY SEQ ID NO: 345 GGDGADIIY SEQ ID NO: 346GNDGADIIY SEQ ID NO: 347 GDDGADIIY SEQ ID NO: 348GGAGNNIIY SEQ ID NO: 349 GNAGNNIIY SEQ ID NO: 350GDAGNNIIY SEQ ID NO: 351 GGSGNNIIY SEQ ID NO: 352GNSGNNIIY SEQ ID NO: 353 GDSGNNIIY SEQ ID NO: 354GGGGNNIIY SEQ ID NO: 355 GNGGNNIIY SEQ ID NO: 356GDGGNNIIY SEQ ID NO: 357 GGDGNNIIY SEQ ID NO: 358GNDGNNIIY SEQ ID NO: 359 GDDGNNIIY SEQ ID NO: 360GGAGDNIIY SEQ ID NO: 361 GNAGDNIIY SEQ ID NO: 362GDAGDNIIY SEQ ID NO: 363 GGSGDNIIY SEQ ID NO: 364GNSGDNIIY SEQ ID NO: 365 GDSGDNIIY SEQ ID NO: 366GGGGDNIIY SEQ ID NO: 367 GNGGDNIIY SEQ ID NO: 368GDGGDNIIY SEQ ID NO: 369 GGDGDNIIY SEQ ID NO: 370GNDGDNIIY SEQ ID NO: 371 GDDGDNIIY SEQ ID NO: 372GGAGANIIY SEQ ID NO: 373 GNAGANIIY SEQ ID NO: 374GDAGANIIY SEQ ID NO: 375 GGSGANIIY SEQ ID NO: 376GNSGANIIY SEQ ID NO: 377 GDSGANIIY SEQ ID NO: 378GGGGANIIY SEQ ID NO: 379 GNGGANIIY SEQ ID NO: 380GDGGANIIY SEQ ID NO: 381 GGDGANIIY SEQ ID NO: 382GNDGANIIY SEQ ID NO: 383 GDDGANIIY SEQ ID NO: 384GGAGNDVIY SEQ ID NO: 385 GNAGNDVIY SEQ ID NO: 386GDAGNDVIY SEQ ID NO: 387 GGSGNDVIY SEQ ID NO: 388GNSGNDVIY SEQ ID NO: 389 GDSGNDVIY SEQ ID NO: 390GGGGNDVIY SEQ ID NO: 391 GNGGNDVIY SEQ ID NO: 392GDGGNDVIY SEQ ID NO: 393 GGDGNDVIY SEQ ID NO: 394GNDGNDVIY SEQ ID NO: 395 GDDGNDVIY SEQ ID NO: 396GGAGDDVIY SEQ ID NO: 397 GNAGDDVIY SEQ ID NO: 398GDAGDDVIY SEQ ID NO: 399 GGSGDDVIY SEQ ID NO: 400GNSGDDVIY SEQ ID NO: 401 GDSGDDVIY SEQ ID NO: 402GGGGDDVIY SEQ ID NO: 403 GNGGDDVIY SEQ ID NO: 404GDGGDDVIY SEQ ID NO: 405 GGDGDDVIY SEQ ID NO: 406GNDGDDVIY SEQ ID NO: 407 GDDGDDVIY SEQ ID NO: 408GGAGADVIY SEQ ID NO: 409 GNAGADVIY SEQ ID NO: 410GDAGADVIY SEQ ID NO: 411 GGSGADVIY SEQ ID NO: 412GNSGADVIY SEQ ID NO: 413 GDSGADVIY SEQ ID NO: 414GGGGADVIY SEQ ID NO: 415 GNGGADVIY SEQ ID NO: 416GDGGADVIY SEQ ID NO: 417 GGDGADVIY SEQ ID NO: 418GNDGADVIY SEQ ID NO: 419 GDDGADVIY SEQ ID NO: 420GGAGNNVIY SEQ ID NO: 421 GNAGNNVIY SEQ ID NO: 422GDAGNNVIY SEQ ID NO: 423 GGSGNNVIY SEQ ID NO: 424GNSGNNVIY SEQ ID NO: 425 GDSGNNVIY SEQ ID NO: 426GGGGNNVIY SEQ ID NO: 427 GNGGNNVIY SEQ ID NO: 428GDGGNNVIY SEQ ID NO: 429 GGDGNNVIY SEQ ID NO: 430GNDGNNVIY SEQ ID NO: 431 GDDGNNVIY SEQ ID NO: 432GGAGDNVIY SEQ ID NO: 433 GNAGDNVIY SEQ ID NO: 434GDAGDNVIY SEQ ID NO: 435 GGSGDNVIY SEQ ID NO: 436GNSGDNVIY SEQ ID NO: 437 GDSGDNVIY SEQ ID NO: 438GGGGDNVIY SEQ ID NO: 439 GNGGDNVIY SEQ ID NO: 440GDGGDNVIY SEQ ID NO: 441 GGDGDNVIY SEQ ID NO: 442GNDGDNVIY SEQ ID NO: 443 GDDGDNVIY SEQ ID NO: 444GGAGANVIY SEQ ID NO: 445 GNAGANVIY SEQ ID NO: 446GDAGANVIY SEQ ID NO: 447 GGSGANVIY SEQ ID NO: 448GNSGANVIY SEQ ID NO: 449 GDSGANVIY SEQ ID NO: 450GGGGANVIY SEQ ID NO: 451 GNGGANVIY SEQ ID NO: 452GDGGANVIY SEQ ID NO: 453 GGDGANVIY SEQ ID NO: 454GNDGANVIY SEQ ID NO: 455 GDDGANVIY SEQ ID NO: 456GGAGNDTLI SEQ ID NO: 457 GNAGNDTLI SEQ ID NO: 458GDAGNDTLI SEQ ID NO: 459 GGSGNDTLI SEQ ID NO: 460GNSGNDTLI SEQ ID NO: 461 GDSGNDTLI SEQ ID NO: 462GGGGNDTLI SEQ ID NO: 463 GNGGNDTLI SEQ ID NO: 464GDGGNDTLI SEQ ID NO: 465 GGDGNDTLI SEQ ID NO: 466GNDGNDTLI SEQ ID NO: 467 GDDGNDTLI SEQ ID NO: 468GGAGDDTLI SEQ ID NO: 469 GNAGDDTLI SEQ ID NO: 470GDAGDDTLI SEQ ID NO: 471 GGSGDDTLI SEQ ID NO: 472GNSGDDTLI SEQ ID NO: 473 GDSGDDTLI SEQ ID NO: 474GGGGDDTLI SEQ ID NO: 475 GNGGDDTLI SEQ ID NO: 476GDGGDDTLI SEQ ID NO: 477 GGDGDDTLI SEQ ID NO: 478GNDGDDTLI SEQ ID NO: 479 GDDGDDTLI SEQ ID NO: 480GGAGADTLI SEQ ID NO: 481 GNAGADTLI SEQ ID NO: 482GDAGADTLI SEQ ID NO: 483 GGSGADTLI SEQ ID NO: 484GNSGADTLI SEQ ID NO: 485 GDSGADTLI SEQ ID NO: 486GGGGADTLI SEQ ID NO: 487 GNGGADTLI SEQ ID NO: 488GDGGADTLI SEQ ID NO: 489 GGDGADTLI SEQ ID NO: 490GNDGADTLI SEQ ID NO: 491 GDDGADTLI SEQ ID NO: 492GGAGNNTLI SEQ ID NO: 493 GNAGNNTLI SEQ ID NO: 494GDAGNNTLI SEQ ID NO: 495 GGSGNNTLI SEQ ID NO: 496GNSGNNTLI SEQ ID NO: 497 GDSGNNTLI SEQ ID NO: 498GGGGNNTLI SEQ ID NO: 499 GNGGNNTLI SEQ ID NO: 500GDGGNNTLI SEQ ID NO: 501 GGDGNNTLI SEQ ID NO: 502GNDGNNTLI SEQ ID NO: 503 GDDGNNTLI SEQ ID NO: 504GGAGDNTLI SEQ ID NO: 505 GNAGDNTLI SEQ ID NO: 506GDAGDNTLI SEQ ID NO: 507 GGSGDNTLI SEQ ID NO: 508GNSGDNTLI SEQ ID NO: 509 GDSGDNTLI SEQ ID NO: 510GGGGDNTLI SEQ ID NO: 511 GNGGDNTLI SEQ ID NO: 512GDGGDNTLI SEQ ID NO: 513 GGDGDNTLI SEQ ID NO: 514GNDGDNTLI SEQ ID NO: 515 GDDGDNTLI SEQ ID NO: 516GGAGANTLI SEQ ID NO: 517 GNAGANTLI SEQ ID NO: 518GDAGANTLI SEQ ID NO: 519 GGSGANTLI SEQ ID NO: 520GNSGANTLI SEQ ID NO: 521 GDSGANTLI SEQ ID NO: 522GGGGANTLI SEQ ID NO: 523 GNGGANTLI SEQ ID NO: 524GDGGANTLI SEQ ID NO: 525 GGDGANTLI SEQ ID NO: 526GNDGANTLI SEQ ID NO: 527 GDDGANTLI SEQ ID NO: 528GGAGNDILI SEQ ID NO: 529 GNAGNDILI SEQ ID NO: 530GDAGNDILI SEQ ID NO: 531 GGSGNDILI SEQ ID NO: 532GNSGNDILI SEQ ID NO: 533 GDSGNDILI SEQ ID NO: 534GGGGNDILI SEQ ID NO: 535 GNGGNDILI SEQ ID NO: 536GDGGNDILI SEQ ID NO: 537 GGDGNDILI SEQ ID NO: 538GNDGNDILI SEQ ID NO: 539 GDDGNDILI SEQ ID NO: 540GGAGDDILI SEQ ID NO: 541 GNAGDDILI SEQ ID NO: 542GDAGDDILI SEQ ID NO: 543 GGSGDDILI SEQ ID NO: 544GNSGDDILI SEQ ID NO: 545 GDSGDDILI SEQ ID NO: 546GGGGDDILI SEQ ID NO: 547 GNGGDDILI SEQ ID NO: 548GDGGDDILI SEQ ID NO: 549 GGDGDDILI SEQ ID NO: 550GNDGDDILI SEQ ID NO: 551 GDDGDDILI SEQ ID NO: 552GGAGADILI SEQ ID NO: 553 GNAGADILI SEQ ID NO: 554GDAGADILI SEQ ID NO: 555 GGSGADILI SEQ ID NO: 556GNSGADILI SEQ ID NO: 557 GDSGADILI SEQ ID NO: 558GGGGADILI SEQ ID NO: 559 GNGGADILI SEQ ID NO: 560GDGGADILI SEQ ID NO: 561 GGDGADILI SEQ ID NO: 562GNDGADILI SEQ ID NO: 563 GDDGADILI SEQ ID NO: 564GGAGNNILI SEQ ID NO: 565 GNAGNNILI SEQ ID NO: 566GDAGNNILI SEQ ID NO: 567 GGSGNNILI SEQ ID NO: 568GNSGNNILI SEQ ID NO: 569 GDSGNNILI SEQ ID NO: 570GGGGNNILI SEQ ID NO: 571 GNGGNNILI SEQ ID NO: 572GDGGNNILI SEQ ID NO: 573 GGDGNNILI SEQ ID NO: 574GNDGNNILI SEQ ID NO: 575 GDDGNNILI SEQ ID NO: 576GGAGDNILI SEQ ID NO: 577 GNAGDNILI SEQ ID NO: 578GDAGDNILI SEQ ID NO: 579 GGSGDNILI SEQ ID NO: 580GNSGDNILI SEQ ID NO: 581 GDSGDNILI SEQ ID NO: 582GGGGDNILI SEQ ID NO: 583 GNGGDNILI SEQ ID NO: 584GDGGDNILI SEQ ID NO: 585 GGDGDNILI SEQ ID NO: 586GNDGDNILI SEQ ID NO: 587 GDDGDNILI SEQ ID NO: 588GGAGANILI SEQ ID NO: 589 GNAGANILI SEQ ID NO: 590GDAGANILI SEQ ID NO: 591 GGSGANILI SEQ ID NO: 592GNSGANILI SEQ ID NO: 593 GDSGANILI SEQ ID NO: 594GGGGANILI SEQ ID NO: 595 GNGGANILI SEQ ID NO: 596GDGGANILI SEQ ID NO: 597 GGDGANILI SEQ ID NO: 598GNDGANILI SEQ ID NO: 599 GDDGANILI SEQ ID NO: 600GGAGNDVLI SEQ ID NO: 601 GNAGNDVLI SEQ ID NO: 602GDAGNDVLI SEQ ID NO: 603 GGSGNDVLI SEQ ID NO: 604GNSGNDVLI SEQ ID NO: 605 GDSGNDVLI SEQ ID NO: 606GGGGNDVLI SEQ ID NO: 607 GNGGNDVLI SEQ ID NO: 608GDGGNDVLI SEQ ID NO: 609 GGDGNDVLI SEQ ID NO: 610GNDGNDVLI SEQ ID NO: 611 GDDGNDVLI SEQ ID NO: 612GGAGDDVLI SEQ ID NO: 613 GNAGDDVLI SEQ ID NO: 614GDAGDDVLI SEQ ID NO: 615 GGSGDDVLI SEQ ID NO: 616GNSGDDVLI SEQ ID NO: 617 GDSGDDVLI SEQ ID NO: 618GGGGDDVLI SEQ ID NO: 619 GNGGDDVLI SEQ ID NO: 620GDGGDDVLI SEQ ID NO: 621 GGDGDDVLI SEQ ID NO: 622GNDGDDVLI SEQ ID NO: 623 GDDGDDVLI SEQ ID NO: 624GGAGADVLI SEQ ID NO: 625 GNAGADVLI SEQ ID NO: 626GDAGADVLI SEQ ID NO: 627 GGSGADVLI SEQ ID NO: 628GNSGADVLI SEQ ID NO: 629 GDSGADVLI SEQ ID NO: 630GGGGADVLI SEQ ID NO: 631 GNGGADVLI SEQ ID NO: 632GDGGADVLI SEQ ID NO: 633 GGDGADVLI SEQ ID NO: 634GNDGADVLI SEQ ID NO: 635 GDDGADVLI SEQ ID NO: 636GGAGNNVLI SEQ ID NO: 637 GNAGNNVLI SEQ ID NO: 638GDAGNNVLI SEQ ID NO: 639 GGSGNNVLI SEQ ID NO: 640GNSGNNVLI SEQ ID NO: 641 GDSGNNVLI SEQ ID NO: 642GGGGNNVLI SEQ ID NO: 643 GNGGNNVLI SEQ ID NO: 644GDGGNNVLI SEQ ID NO: 645 GGDGNNVLI SEQ ID NO: 646GNDGNNVLI SEQ ID NO: 647 GDDGNNVLI SEQ ID NO: 648GGAGDNVLI SEQ ID NO: 649 GNAGDNVLI SEQ ID NO: 650GDAGDNVLI SEQ ID NO: 651 GGSGDNVLI SEQ ID NO: 652GNSGDNVLI SEQ ID NO: 653 GDSGDNVLI SEQ ID NO: 654GGGGDNVLI SEQ ID NO: 655 GNGGDNVLI SEQ ID NO: 656GDGGDNVLI SEQ ID NO: 657 GGDGDNVLI SEQ ID NO: 658GNDGDNVLI SEQ ID NO: 659 GDDGDNVLI SEQ ID NO: 660GGAGANVLI SEQ ID NO: 661 GNAGANVLI SEQ ID NO: 662GDAGANVLI SEQ ID NO: 663 GGSGANVLI SEQ ID NO: 664GNSGANVLI SEQ ID NO: 665 GDSGANVLI SEQ ID NO: 666GGGGANVLI SEQ ID NO: 667 GNGGANVLI SEQ ID NO: 668GDGGANVLI SEQ ID NO: 669 GGDGANVLI SEQ ID NO: 670GNDGANVLI SEQ ID NO: 671 GDDGANVLI SEQ ID NO: 672GGAGNDTII SEQ ID NO: 673 GNAGNDTII SEQ ID NO: 674GDAGNDTII SEQ ID NO: 675 GGSGNDTII SEQ ID NO: 676GNSGNDTII SEQ ID NO: 677 GDSGNDTII SEQ ID NO: 678GGGGNDTII SEQ ID NO: 679 GNGGNDTII SEQ ID NO: 680GDGGNDTII SEQ ID NO: 681 GGDGNDTII SEQ ID NO: 682GNDGNDTII SEQ ID NO: 683 GDDGNDTII SEQ ID NO: 684GGAGDDTII SEQ ID NO: 685 GNAGDDTII SEQ ID NO: 686GDAGDDTII SEQ ID NO: 687 GGSGDDTII SEQ ID NO: 688GNSGDDTII SEQ ID NO: 689 GDSGDDTII SEQ ID NO: 690GGGGDDTII SEQ ID NO: 691 GNGGDDTII SEQ ID NO: 692GDGGDDTII SEQ ID NO: 693 GGDGDDTII SEQ ID NO: 694GNDGDDTII SEQ ID NO: 695 GDDGDDTII SEQ ID NO: 696GGAGADTII SEQ ID NO: 697 GNAGADTII SEQ ID NO: 698GDAGADTII SEQ ID NO: 699 GGSGADTII SEQ ID NO: 700GNSGADTII SEQ ID NO: 701 GDSGADTII SEQ ID NO: 702GGGGADTII SEQ ID NO: 703 GNGGADTII SEQ ID NO: 704GDGGADTII SEQ ID NO: 705 GGDGADTII SEQ ID NO: 706GNDGADTII SEQ ID NO: 707 GDDGADTII SEQ ID NO: 708GGAGNNTII SEQ ID NO: 709 GNAGNNTII SEQ ID NO: 710GDAGNNTII SEQ ID NO: 711 GGSGNNTII SEQ ID NO: 712GNSGNNTII SEQ ID NO: 713 GDSGNNTII SEQ ID NO: 714GGGGNNTII SEQ ID NO: 715 GNGGNNTII SEQ ID NO: 716GDGGNNTII SEQ ID NO: 717 GGDGNNTII SEQ ID NO: 718GNDGNNTII SEQ ID NO: 719 GDDGNNTII SEQ ID NO: 720GGAGDNTII SEQ ID NO: 721 GNAGDNTII SEQ ID NO: 722GDAGDNTII SEQ ID NO: 723 GGSGDNTII SEQ ID NO: 724GNSGDNTII SEQ ID NO: 725 GDSGDNTII SEQ ID NO: 726GGGGDNTII SEQ ID NO: 727 GNGGDNTII SEQ ID NO: 728GDGGDNTII SEQ ID NO: 729 GGDGDNTII SEQ ID NO: 730GNDGDNTII SEQ ID NO: 731 GDDGDNTII SEQ ID NO: 732GGAGANTII SEQ ID NO: 733 GNAGANTII SEQ ID NO: 734GDAGANTII SEQ ID NO: 735 GGSGANTII SEQ ID NO: 736GNSGANTII SEQ ID NO: 737 GDSGANTII SEQ ID NO: 738GGGGANTII SEQ ID NO: 739 GNGGANTII SEQ ID NO: 740GDGGANTII SEQ ID NO: 741 GGDGANTII SEQ ID NO: 742GNDGANTII SEQ ID NO: 743 GDDGANTII SEQ ID NO: 744GGAGNDIII SEQ ID NO: 745 GNAGNDIII SEQ ID NO: 746GDAGNDIII SEQ ID NO: 747 GGSGNDIII SEQ ID NO: 748GNSGNDIII SEQ ID NO: 749 GDSGNDIII SEQ ID NO: 750GGGGNDIII SEQ ID NO: 751 GNGGNDIII SEQ ID NO: 752GDGGNDIII SEQ ID NO: 753 GGDGNDIII SEQ ID NO: 754GNDGNDIII SEQ ID NO: 755 GDDGNDIII SEQ ID NO: 756GGAGDDIII SEQ ID NO: 757 GNAGDDIII SEQ ID NO: 758GDAGDDIII SEQ ID NO: 759 GGSGDDIII SEQ ID NO: 760GNSGDDIII SEQ ID NO: 761 GDSGDDIII SEQ ID NO: 762GGGGDDIII SEQ ID NO: 763 GNGGDDIII SEQ ID NO: 764GDGGDDIII SEQ ID NO: 765 GGDGDDIII SEQ ID NO: 766GNDGDDIII SEQ ID NO: 767 GDDGDDIII SEQ ID NO: 768GGAGADIII SEQ ID NO: 769 GNAGADIII SEQ ID NO: 770GDAGADIII SEQ ID NO: 771 GGSGADIII SEQ ID NO: 772GNSGADIII SEQ ID NO: 773 GDSGADIII SEQ ID NO: 774GGGGADIII SEQ ID NO: 775 GNGGADIII SEQ ID NO: 776GDGGADIII SEQ ID NO: 777 GGDGADIII SEQ ID NO: 778GNDGADIII SEQ ID NO: 779 GDDGADIII SEQ ID NO: 780GGAGNNIII SEQ ID NO: 781 GNAGNNIII SEQ ID NO: 782GDAGNNIII SEQ ID NO: 783 GGSGNNIII SEQ ID NO: 784GNSGNNIII SEQ ID NO: 785 GDSGNNIII SEQ ID NO: 786GGGGNNIII SEQ ID NO: 787 GNGGNNIII SEQ ID NO: 788GDGGNNIII SEQ ID NO: 789 GGDGNNIII SEQ ID NO: 790GNDGNNIII SEQ ID NO: 791 GDDGNNIII SEQ ID NO: 792GGAGDNIII SEQ ID NO: 793 GNAGDNIII SEQ ID NO: 794GDAGDNIII SEQ ID NO: 795 GGSGDNIII SEQ ID NO: 796GNSGDNIII SEQ ID NO: 797 GDSGDNIII SEQ ID NO: 798GGGGDNIII SEQ ID NO: 799 GNGGDNIII SEQ ID NO: 800GDGGDNIII SEQ ID NO: 801 GGDGDNIII SEQ ID NO: 802GNDGDNIII SEQ ID NO: 803 GDDGDNIII SEQ ID NO: 804GGAGANIII SEQ ID NO: 805 GNAGANIII SEQ ID NO: 806GDAGANIII SEQ ID NO: 807 GGSGANIII SEQ ID NO: 808GNSGANIII SEQ ID NO: 809 GDSGANIII SEQ ID NO: 810GGGGANIII SEQ ID NO: 811 GNGGANIII SEQ ID NO: 812GDGGANIII SEQ ID NO: 813 GGDGANIII SEQ ID NO: 814GNDGANIII SEQ ID NO: 815 GDDGANIII SEQ ID NO: 816GGAGNDVII SEQ ID NO: 817 GNAGNDVII SEQ ID NO: 818GDAGNDVII SEQ ID NO: 819 GGSGNDVII SEQ ID NO: 820GNSGNDVII SEQ ID NO: 821 GDSGNDVII SEQ ID NO: 822GGGGNDVII SEQ ID NO: 823 GNGGNDVII SEQ ID NO: 824GDGGNDVII SEQ ID NO: 825 GGDGNDVII SEQ ID NO: 826GNDGNDVII SEQ ID NO: 827 GDDGNDVII SEQ ID NO: 828GGAGDDVII SEQ ID NO: 829 GNAGDDVII SEQ ID NO: 830GDAGDDVII SEQ ID NO: 831 GGSGDDVII SEQ ID NO: 832GNSGDDVII SEQ ID NO: 833 GDSGDDVII SEQ ID NO: 834GGGGDDVII SEQ ID NO: 835 GNGGDDVII SEQ ID NO: 836GDGGDDVII SEQ ID NO: 837 GGDGDDVII SEQ ID NO: 838GNDGDDVII SEQ ID NO: 839 GDDGDDVII SEQ ID NO: 840GGAGADVII SEQ ID NO: 841 GNAGADVII SEQ ID NO: 842GDAGADVII SEQ ID NO: 843 GGSGADVII SEQ ID NO: 844GNSGADVII SEQ ID NO: 845 GDSGADVII SEQ ID NO: 846GGGGADVII SEQ ID NO: 847 GNGGADVII SEQ ID NO: 848GDGGADVII SEQ ID NO: 849 GGDGADVII SEQ ID NO: 850GNDGADVII SEQ ID NO: 851 GDDGADVII SEQ ID NO: 852GGAGNNVII SEQ ID NO: 853 GNAGNNVII SEQ ID NO: 854GDAGNNVII SEQ ID NO: 855 GGSGNNVII SEQ ID NO: 856GNSGNNVII SEQ ID NO: 857 GDSGNNVII SEQ ID NO: 858GGGGNNVII SEQ ID NO: 859 GNGGNNVII SEQ ID NO: 860GDGGNNVII SEQ ID NO: 861 GGDGNNVII SEQ ID NO: 862GNDGNNVII SEQ ID NO: 863 GDDGNNVII SEQ ID NO: 864GGAGDNVII SEQ ID NO: 865 GNAGDNVII SEQ ID NO: 866GDAGDNVII SEQ ID NO: 867 GGSGDNVII SEQ ID NO: 868GNSGDNVII SEQ ID NO: 869 GDSGDNVII SEQ ID NO: 870GGGGDNVII SEQ ID NO: 871 GNGGDNVII SEQ ID NO: 872GDGGDNVII SEQ ID NO: 873 GGDGDNVII SEQ ID NO: 874GNDGDNVII SEQ ID NO: 875 GDDGDNVII SEQ ID NO: 876GGAGANVII SEQ ID NO: 877 GNAGANVII SEQ ID NO: 878GDAGANVII SEQ ID NO: 879 GGSGANVII SEQ ID NO: 880GNSGANVII SEQ ID NO: 881 GDSGANVII SEQ ID NO: 882GGGGANVII SEQ ID NO: 883 GNGGANVII SEQ ID NO: 884GDGGANVII SEQ ID NO: 885 GGDGANVII SEQ ID NO: 886GNDGANVII SEQ ID NO: 887 GDDGANVII SEQ ID NO: 888GGAGNDTLV SEQ ID NO: 889 GNAGNDTLV SEQ ID NO: 890GDAGNDTLV SEQ ID NO: 891 GGSGNDTLV SEQ ID NO: 892GNSGNDTLV SEQ ID NO: 893 GDSGNDTLV SEQ ID NO: 894GGGGNDTLV SEQ ID NO: 895 GNGGNDTLV SEQ ID NO: 896GDGGNDTLV SEQ ID NO: 897 GGDGNDTLV SEQ ID NO: 898GNDGNDTLV SEQ ID NO: 899 GDDGNDTLV SEQ ID NO: 900GGAGDDTLV SEQ ID NO: 901 GNAGDDTLV SEQ ID NO: 902GDAGDDTLV SEQ ID NO: 903 GGSGDDTLV SEQ ID NO: 904GNSGDDTLV SEQ ID NO: 905 GDSGDDTLV SEQ ID NO: 906GGGGDDTLV SEQ ID NO: 907 GNGGDDTLV SEQ ID NO: 908GDGGDDTLV SEQ ID NO: 909 GGDGDDTLV SEQ ID NO: 910GNDGDDTLV SEQ ID NO: 911 GDDGDDTLV SEQ ID NO: 912GGAGADTLV SEQ ID NO: 913 GNAGADTLV SEQ ID NO: 914GDAGADTLV SEQ ID NO: 915 GGSGADTLV SEQ ID NO: 916GNSGADTLV SEQ ID NO: 917 GDSGADTLV SEQ ID NO: 918GGGGADTLV SEQ ID NO: 919 GNGGADTLV SEQ ID NO: 920GDGGADTLV SEQ ID NO: 921 GGDGADTLV SEQ ID NO: 922GNDGADTLV SEQ ID NO: 923 GDDGADTLV SEQ ID NO: 924GGAGNNTLV SEQ ID NO: 925 GNAGNNTLV SEQ ID NO: 926GDAGNNTLV SEQ ID NO: 927 GGSGNNTLV SEQ ID NO: 928GNSGNNTLV SEQ ID NO: 929 GDSGNNTLV SEQ ID NO: 930GGGGNNTLV SEQ ID NO: 931 GNGGNNTLV SEQ ID NO: 932GDGGNNTLV SEQ ID NO: 933 GGDGNNTLV SEQ ID NO: 934GNDGNNTLV SEQ ID NO: 935 GDDGNNTLV SEQ ID NO: 936GGAGDNTLV SEQ ID NO: 937 GNAGDNTLV SEQ ID NO: 938GDAGDNTLV SEQ ID NO: 939 GGSGDNTLV SEQ ID NO: 940GNSGDNTLV SEQ ID NO: 941 GDSGDNTLV SEQ ID NO: 942GGGGDNTLV SEQ ID NO: 943 GNGGDNTLV SEQ ID NO: 944GDGGDNTLV SEQ ID NO: 945 GGDGDNTLV SEQ ID NO: 946GNDGDNTLV SEQ ID NO: 947 GDDGDNTLV SEQ ID NO: 948GGAGANTLV SEQ ID NO: 949 GNAGANTLV SEQ ID NO: 950GDAGANTLV SEQ ID NO: 951 GGSGANTLV SEQ ID NO: 952GNSGANTLV SEQ ID NO: 953 GDSGANTLV SEQ ID NO: 954GGGGANTLV SEQ ID NO: 955 GNGGANTLV SEQ ID NO: 956GDGGANTLV SEQ ID NO: 957 GGDGANTLV SEQ ID NO: 958GNDGANTLV SEQ ID NO: 959 GDDGANTLV SEQ ID NO: 960GGAGNDILV SEQ ID NO: 961 GNAGNDILV SEQ ID NO: 962GDAGNDILV SEQ ID NO: 963 GGSGNDILV SEQ ID NO: 964GNSGNDILV SEQ ID NO: 965 GDSGNDILV SEQ ID NO: 966GGGGNDILV SEQ ID NO: 967 GNGGNDILV SEQ ID NO: 968GDGGNDILV SEQ ID NO: 969 GGDGNDILV SEQ ID NO: 970GNDGNDILV SEQ ID NO: 971 GDDGNDILV SEQ ID NO: 972GGAGDDILV SEQ ID NO: 973 GNAGDDILV SEQ ID NO: 974GDAGDDILV SEQ ID NO: 975 GGSGDDILV SEQ ID NO: 976GNSGDDILV SEQ ID NO: 977 GDSGDDILV SEQ ID NO: 978GGGGDDILV SEQ ID NO: 979 GNGGDDILV SEQ ID NO: 980GDGGDDILV SEQ ID NO: 981 GGDGDDILV SEQ ID NO: 982GNDGDDILV SEQ ID NO: 983 GDDGDDILV SEQ ID NO: 984GGAGADILV SEQ ID NO: 985 GNAGADILV SEQ ID NO: 986GDAGADILV SEQ ID NO: 987 GGSGADILV SEQ ID NO: 988GNSGADILV SEQ ID NO: 989 GDSGADILV SEQ ID NO: 990GGGGADILV SEQ ID NO: 991 GNGGADILV SEQ ID NO: 992GDGGADILV SEQ ID NO: 993 GGDGADILV SEQ ID NO: 994GNDGADILV SEQ ID NO: 995 GDDGADILV SEQ ID NO: 996GGAGNNILV SEQ ID NO: 997 GNAGNNILV SEQ ID NO: 998GDAGNNILV SEQ ID NO: 999 GGSGNNILV SEQ ID NO: 1000GNSGNNILV SEQ ID NO: 1001 GDSGNNILV SEQ ID NO: 1002GGGGNNILV SEQ ID NO: 1003 GNGGNNILV SEQ ID NO: 1004GDGGNNILV SEQ ID NO: 1005 GGDGNNILV SEQ ID NO: 1006GNDGNNILV SEQ ID NO: 1007 GDDGNNILV SEQ ID NO: 1008GGAGDNILV SEQ ID NO: 1009 GNAGDNILV SEQ ID NO: 1010GDAGDNILV SEQ ID NO: 1011 GGSGDNILV SEQ ID NO: 1012GNSGDNILV SEQ ID NO: 1013 GDSGDNILV SEQ ID NO: 1014GGGGDNILV SEQ ID NO: 1015 GNGGDNILV SEQ ID NO: 1016GDGGDNILV SEQ ID NO: 1017 GGDGDNILV SEQ ID NO: 1018GNDGDNILV SEQ ID NO: 1019 GDDGDNILV SEQ ID NO: 1020GGAGANILV SEQ ID NO: 1021 GNAGANILV SEQ ID NO: 1022GDAGANILV SEQ ID NO: 1023 GGSGANILV SEQ ID NO: 1024GNSGANILV SEQ ID NO: 1025 GDSGANILV SEQ ID NO: 1026GGGGANILV SEQ ID NO: 1027 GNGGANILV SEQ ID NO: 1028GDGGANILV SEQ ID NO: 1029 GGDGANILV SEQ ID NO: 1030GNDGANILV SEQ ID NO: 1031 GDDGANILV SEQ ID NO: 1032GGAGNDVLV SEQ ID NO: 1033 GNAGNDVLV SEQ ID NO: 1034GDAGNDVLV SEQ ID NO: 1035 GGSGNDVLV SEQ ID NO: 1036GNSGNDVLV SEQ ID NO: 1037 GDSGNDVLV SEQ ID NO: 1038GGGGNDVLV SEQ ID NO: 1039 GNGGNDVLV SEQ ID NO: 1040GDGGNDVLV SEQ ID NO: 1041 GGDGNDVLV SEQ ID NO: 1042GNDGNDVLV SEQ ID NO: 1043 GDDGNDVLV SEQ ID NO: 1044GGAGDDVLV SEQ ID NO: 1045 GNAGDDVLV SEQ ID NO: 1046GDAGDDVLV SEQ ID NO: 1047 GGSGDDVLV SEQ ID NO: 1048GNSGDDVLV SEQ ID NO: 1049 GDSGDDVLV SEQ ID NO: 1050GGGGDDVLV SEQ ID NO: 1051 GNGGDDVLV SEQ ID NO: 1052GDGGDDVLV SEQ ID NO: 1053 GGDGDDVLV SEQ ID NO: 1054GNDGDDVLV SEQ ID NO: 1055 GDDGDDVLV SEQ ID NO: 1056GGAGADVLV SEQ ID NO: 1057 GNAGADVLV SEQ ID NO: 1058GDAGADVLV SEQ ID NO: 1059 GGSGADVLV SEQ ID NO: 1060GNSGADVLV SEQ ID NO: 1061 GDSGADVLV SEQ ID NO: 1062GGGGADVLV SEQ ID NO: 1063 GNGGADVLV SEQ ID NO: 1064GDGGADVLV SEQ ID NO: 1065 GGDGADVLV SEQ ID NO: 1066GNDGADVLV SEQ ID NO: 1067 GDDGADVLV SEQ ID NO: 1068GGAGNNVLV SEQ ID NO: 1069 GNAGNNVLV SEQ ID NO: 1070GDAGNNVLV SEQ ID NO: 1071 GGSGNNVLV SEQ ID NO: 1072GNSGNNVLV SEQ ID NO: 1073 GDSGNNVLV SEQ ID NO: 1074GGGGNNVLV SEQ ID NO: 1075 GNGGNNVLV SEQ ID NO: 1076GDGGNNVLV SEQ ID NO: 1077 GGDGNNVLV SEQ ID NO: 1078GNDGNNVLV SEQ ID NO: 1079 GDDGNNVLV SEQ ID NO: 1080GGAGDNVLV SEQ ID NO: 1081 GNAGDNVLV SEQ ID NO: 1082GDAGDNVLV SEQ ID NO: 1083 GGSGDNVLV SEQ ID NO: 1084GNSGDNVLV SEQ ID NO: 1085 GDSGDNVLV SEQ ID NO: 1086GGGGDNVLV SEQ ID NO: 1087 GNGGDNVLV SEQ ID NO: 1088GDGGDNVLV SEQ ID NO: 1089 GGDGDNVLV SEQ ID NO: 1090GNDGDNVLV SEQ ID NO: 1091 GDDGDNVLV SEQ ID NO: 1092GGAGANVLV SEQ ID NO: 1093 GNAGANVLV SEQ ID NO: 1094GDAGANVLV SEQ ID NO: 1095 GGSGANVLV SEQ ID NO: 1096GNSGANVLV SEQ ID NO: 1097 GDSGANVLV SEQ ID NO: 1098GGGGANVLV SEQ ID NO: 1099 GNGGANVLV SEQ ID NO: 1100GDGGANVLV SEQ ID NO: 1101 GGDGANVLV SEQ ID NO: 1102GNDGANVLV SEQ ID NO: 1103 GDDGANVLV SEQ ID NO: 1104GGAGNDTIV SEQ ID NO: 1105 GNAGNDTIV SEQ ID NO: 1106GDAGNDTIV SEQ ID NO: 1107 GGSGNDTIV SEQ ID NO: 1108GNSGNDTIV SEQ ID NO: 1109 GDSGNDTIV SEQ ID NO: 1110GGGGNDTIV SEQ ID NO: 1111 GNGGNDTIV SEQ ID NO: 1112GDGGNDTIV SEQ ID NO: 1113 GGDGNDTIV SEQ ID NO: 1114GNDGNDTIV SEQ ID NO: 1115 GDDGNDTIV SEQ ID NO: 1116GGAGDDTIV SEQ ID NO: 1117 GNAGDDTIV SEQ ID NO: 1118GDAGDDTIV SEQ ID NO: 1119 GGSGDDTIV SEQ ID NO: 1120GNSGDDTIV SEQ ID NO: 1121 GDSGDDTIV SEQ ID NO: 1122GGGGDDTIV SEQ ID NO: 1123 GNGGDDTIV SEQ ID NO: 1124GDGGDDTIV SEQ ID NO: 1125 GGDGDDTIV SEQ ID NO: 1126GNDGDDTIV SEQ ID NO: 1127 GDDGDDTIV SEQ ID NO: 1128GGAGADTIV SEQ ID NO: 1129 GNAGADTIV SEQ ID NO: 1130GDAGADTIV SEQ ID NO: 1131 GGSGADTIV SEQ ID NO: 1132GNSGADTIV SEQ ID NO: 1133 GDSGADTIV SEQ ID NO: 1134GGGGADTIV SEQ ID NO: 1135 GNGGADTIV SEQ ID NO: 1136GDGGADTIV SEQ ID NO: 1137 GGDGADTIV SEQ ID NO: 1138GNDGADTIV SEQ ID NO: 1139 GDDGADTIV SEQ ID NO: 1140GGAGNNTIV SEQ ID NO: 1141 GNAGNNTIV SEQ ID NO: 1142GDAGNNTIV SEQ ID NO: 1143 GGSGNNTIV SEQ ID NO: 1144GNAGNNTIV SEQ ID NO: 1145 GDSGNNTIV SEQ ID NO: 1146GGGGNNTIV SEQ ID NO: 1147 GNGGNNTIV SEQ ID NO: 1148GDGGNNTIV SEQ ID NO: 1149 GGDGNNTIV SEQ ID NO: 1150GNDGNNTIV SEQ ID NO: 1151 GDDGNNTIV SEQ ID NO: 1152GGAGDNTIV SEQ ID NO: 1153 GNAGDNTIV SEQ ID NO: 1154GDAGDNTIV SEQ ID NO: 1155 GGSGDNTIV SEQ ID NO: 1156GNSGDNTIV SEQ ID NO: 1157 GDSGDNTIV SEQ ID NO: 1158GGGGDNTIV SEQ ID NO: 1159 GNGGDNTIV SEQ ID NO: 1160GDGGDNTIV SEQ ID NO: 1161 GGDGDNTIV SEQ ID NO: 1162GNDGDNTIV SEQ ID NO: 1163 GDDGDNTIV SEQ ID NO: 1164GGAGANTIV SEQ ID NO: 1165 GNAGANTIV SEQ ID NO: 1166GDAGANTIV SEQ ID NO: 1167 GGSGANTIV SEQ ID NO: 1168GNSGANTIV SEQ ID NO: 1169 GDSGANTIV SEQ ID NO: 1170GGGGANTIV SEQ ID NO: 1171 GNGGANTIV SEQ ID NO: 1172GDGGANTIV SEQ ID NO: 1173 GGDGANTIV SEQ ID NO: 1174GNDGANTIV SEQ ID NO: 1175 GDDGANTIV SEQ ID NO: 1176GGAGNDIIV SEQ ID NO: 1177 GNAGNDIIV SEQ ID NO: 1178GDAGNDIIV SEQ ID NO: 1179 GGSGNDIIV SEQ ID NO: 1180GNSGNDIIV SEQ ID NO: 1181 GDSGNDIIV SEQ ID NO: 1182GGGGNDIIV SEQ ID NO: 1183 GNGGNDIIV SEQ ID NO: 1184GDGGNDIIV SEQ ID NO: 1185 GGDGNDIIV SEQ ID NO: 1186GNDGNDIIV SEQ ID NO: 1187 GDDGNDIIV SEQ ID NO: 1188GGAGDDIIV SEQ ID NO: 1189 GNAGDDIIV SEQ ID NO: 1190GDAGDDIIV SEQ ID NO: 1191 GGSGDDIIV SEQ ID NO: 1192GNSGDDIIV SEQ ID NO: 1193 GDSGDDIIV SEQ ID NO: 1194GGGGDDIIV SEQ ID NO: 1195 GNGGDDIIV SEQ ID NO: 1196GDGGDDIIV SEQ ID NO: 1197 GGDGDDIIV SEQ ID NO: 1198GNDGDDIIV SEQ ID NO: 1199 GDDGDDIIV SEQ ID NO: 1200GGAGADIIV SEQ ID NO: 1201 GNAGADIIV SEQ ID NO: 1202GDAGADIIV SEQ ID NO: 1203 GGSGADIIV SEQ ID NO: 1204GNSGADIIV SEQ ID NO: 1205 GDSGADIIV SEQ ID NO: 1206GGGGADIIV SEQ ID NO: 1207 GNGGADIIV SEQ ID NO: 1208GDGGADIIV SEQ ID NO: 1209 GGDGADIIV SEQ ID NO: 1210GNDGADIIV SEQ ID NO: 1211 GDDGADIIV SEQ ID NO: 1212GGAGNNIIV SEQ ID NO: 1213 GNAGNNIIV SEQ ID NO: 1214GDAGNNIIV SEQ ID NO: 1215 GGSGNNIIV SEQ ID NO: 1216GNSGNNIIV SEQ ID NO: 1217 GDSGNNIIV SEQ ID NO: 1218GGGGNNIIV SEQ ID NO: 1219 GNGGNNIIV SEQ ID NO: 1220GDGGNNIIV SEQ ID NO: 1221 GGDGNNIIV SEQ ID NO: 1222GNDGNNIIV SEQ ID NO: 1223 GDDGNNIIV SEQ ID NO: 1224GGAGDNIIV SEQ ID NO: 1225 GNAGDNIIV SEQ ID NO: 1226GDAGDNIIV SEQ ID NO: 1227 GGSGDNIIV SEQ ID NO: 1228GNSGDNIIV SEQ ID NO: 1229 GDSGDNIIV SEQ ID NO: 1230GGGGDNIIV SEQ ID NO: 1231 GNGGDNIIV SEQ ID NO: 1232GDGGDNIIV SEQ ID NO: 1233 GGDGDNIIV SEQ ID NO: 1234GNDGDNIIV SEQ ID NO: 1235 GDDGDNIIV SEQ ID NO: 1236GGAGANIIV SEQ ID NO: 1237 GNAGANIIV SEQ ID NO: 1238GDAGANIIV SEQ ID NO: 1239 GGSGANIIV SEQ ID NO: 1240GNSGANIIV SEQ ID NO: 1241 GDSGANIIV SEQ ID NO: 1242GGGGANIIV SEQ ID NO: 1243 GNGGANIIV SEQ ID NO: 1244GDGGANIIV SEQ ID NO: 1245 GGDGANIIV SEQ ID NO: 1246GNDGANIIV SEQ ID NO: 1247 GDDGANIIV SEQ ID NO: 1248GGAGNDVIV SEQ ID NO: 1249 GNAGNDVIV SEQ ID NO: 1250GDAGNDVIV SEQ ID NO: 1251 GGSGNDVIV SEQ ID NO: 1252GNSGNDVIV SEQ ID NO: 1253 GDSGNDVIV SEQ ID NO: 1254GGGGNDVIV SEQ ID NO: 1255 GNGGNDVIV SEQ ID NO: 1256GDGGNDVIV SEQ ID NO: 1257 GGDGNDVIV SEQ ID NO: 1258GNDGNDVIV SEQ ID NO: 1259 GDDGNDVIV SEQ ID NO: 1260GGAGDDVIV SEQ ID NO: 1261 GNAGDDVIV SEQ ID NO: 1262GDAGDDVIV SEQ ID NO: 1263 GGSGDDVIV SEQ ID NO: 1264GNSGDDVIV SEQ ID NO: 1265 GDSGDDVIV SEQ ID NO: 1266GGGGDDVIV SEQ ID NO: 1267 GNGGDDVIV SEQ ID NO: 1268GDGGDDVIV SEQ ID NO: 1269 GGDGDDVIV SEQ ID NO: 1270GNDGDDVIV SEQ ID NO: 1271 GDDGDDVIV SEQ ID NO: 1272GGAGADVIV SEQ ID NO: 1273 GNAGADVIV SEQ ID NO: 1274GDAGADVIV SEQ ID NO: 1275 GGSGADVIV SEQ ID NO: 1276GNSGADVIV SEQ ID NO: 1277 GDSGADVIV SEQ ID NO: 1278GGGGADVIV SEQ ID NO: 1279 GNGGADVIV SEQ ID NO: 1280GDGGADVIV SEQ ID NO: 1281 GGDGADVIV SEQ ID NO: 1282GNDGADVIV SEQ ID NO: 1283 GDDGADVIV SEQ ID NO: 1284GGAGNNVIV SEQ ID NO: 1285 GNAGNNVIV SEQ ID NO: 1286GDAGNNVIV SEQ ID NO: 1287 GGSGNNVIV SEQ ID NO: 1288GNSGNNVIV SEQ ID NO: 1289 GDSGNNVIV SEQ ID NO: 1290GGGGNNVIV SEQ ID NO: 1291 GNGGNNVIV SEQ ID NO: 1292GDGGNNVIV SEQ ID NO: 1293 GGDGNNVIV SEQ ID NO: 1294GNDGNNVIV SEQ ID NO: 1295 GDDGNNVIV SEQ ID NO: 1296GGAGDNVIV SEQ ID NO: 1297 GNAGDNVIV SEQ ID NO: 1298GDAGDNVIV SEQ ID NO: 1299 GGSGDNVIV SEQ ID NO: 1300GNSGDNVIV SEQ ID NO: 1301 GDSGDNVIV SEQ ID NO: 1302GGGGDNVIV SEQ ID NO: 1303 GNGGDNVIV SEQ ID NO: 1304GDGGDNVIV SEQ ID NO: 1305 GGDGDNVIV SEQ ID NO: 1306GNDGDNVIV SEQ ID NO: 1307 GDDGDNVIV SEQ ID NO: 1308GGAGANVIV SEQ ID NO: 1309 GNAGANVIV SEQ ID NO: 1310GDAGANVIV SEQ ID NO: 1311 GGSGANVIV SEQ ID NO: 1312GNSGANVIV SEQ ID NO: 1313 GDSGANVIV SEQ ID NO: 1314GGGGANVIV SEQ ID NO: 1315 GNGGANVIV SEQ ID NO: 1316GDGGANVIV SEQ ID NO: 1317 GGDGANVIV SEQ ID NO: 1318GNDGANVIV SEQ ID NO: 1319 GDDGANVIV SEQ ID NO: 1320GDEASDLFF SEQ ID NO: 1321 GDLASDLFF SEQ ID NO: 1322GDNASDLFF SEQ ID NO: 1323 GDEASDLFT SEQ ID NO: 1324GDLASDLFT SEQ ID NO: 1325 GDNASDLFT SEQ ID NO: 1326GDEASDLFN SEQ ID NO: 1327 GDLASDLFN SEQ ID NO: 1328GDNASDLFN SEQ ID NO: 1329 GDEASDLFD SEQ ID NO: 1330GDLASDLFD SEQ ID NO: 1331 GDNASDLFD SEQ ID NO: 1332GDEASDLFK SEQ ID NO: 1333 GDLASDLFK SEQ ID NO: 1334GDNASDLFK SEQ ID NO: 1335 GDEASDLFS SEQ ID NO: 1336GDLASDLFS SEQ ID NO: 1337

As used herein, the term “precipitable-beta roll cassette” (PBRC) refersto an amino acid sequence comprising at least one PBRT. In certainembodiments, a PBRC will comprise at least two PBRTs. In certainembodiments, a PBRC will comprise at least 3 PBRTs, at least 4 PBRTs, atleast 5 PBRTs, at least 6 PBRTs, at least 7 PBRTs, at least 8 PBRTs, atleast 9 PBRTs, at least 10 PBRTs, at least 11 PBRTs, at least 12 PBRTs,at least 13 PBRTs, at least 14 PBRTs, at least 15 PBRTs, at least 16PBRTs, at least 17 PBRTs, at least 18 PBRTs, at least 19 PBRTs, at least20 PBRTs, or 20 or more PBRTs. In certain embodiments, the PBRCsdescribed herein will comprise a plurality of precipitable beta rolltags arranged in a tandem repeat. For example, in certain embodiments,the PBRCs described herein can comprise at least 2 PBRTs, at least 3PBRTs, at least 4 PBRTs, at least 5 PBRTs, at least 6 PBRTs, at least 7PBRTs, at least 8 PBRTs, at least 9 PBRTs, at least 10 PBRTs, at least11 PBRTs, at least 12 PBRTs, at least 13 PBRTs, at least 14 PBRTs, atleast 15 PBRTs, at least 16 PBRTs, at least 17 PBRTs, at least 18 PBRTs,at least 19 PBRTs, at least 20 PBRTs, or 20 or more PBRTs in tandemrepeat. In certain embodiments, a PBRC can comprise at least two PBRCsseparated by a linking amino acid sequence. Where a linking amino acidsequence in present between two PBRTs, a PBRT located at either end ofthe linking sequence can be an individual PBRT or it can be a PBRT thatis part of a tandem arrangement of two or more PBRTs.

The PBRCs can comprise polymeric or oligomeric repeats of a PBRT. Incertain embodiments, the PBRCs described herein can comprise one or moredifferent PBRTs. In one embodiment, all of the PBRTs comprised in a PBRCare identical in amino acid sequence. In one embodiment, all of thePBRTs comprised in a PBRC have different amino acid sequences. In oneembodiment, at least one PBRT comprised in a PBRC has a different aminoacid sequence as compared to another PBRT in the PBRC. Thus, in certainembodiments, the PBRCs described herein can comprise at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, at least 27, at least 28, at least 29, orat least 30 different PBRTs.

In certain embodiments, a PBRC can also comprise a capping sequence(“CS”) refers to the an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 3. The term “capping sequence” also refers to acapping sequence having the amino acid sequence of any of SEQ ID NO:4-23.

Without wishing to be bound to theory, in some embodiments of theinvention, the ability of polypeptide comprising one or more PBRT toundergo reversible Ca2+ precipitation, can require that the one or morePBRTs be located N-terminally or C-terminally to a capping sequence.Thus, in certain embodiments, the capping sequence is an amino acidsequence, which, when located C-terminally or N-terminally to one ormore PBRTs, allows the one or more PBRTs bind to Ca2+, undergoreversible precipitation in the presence of Ca2+, or induce reversibleprecipitation of a purification moiety linked to the PBRC.

Thus in certain embodiments, a PBRC will comprise at least 2 PBRTs, atleast 3 PBRTs, at least 4 PBRTs, at least 5 PBRTs, at least 6 PBRTs, atleast 7 PBRTs, at least 8 PBRTs, at least 9 PBRTs, at least 10 PBRTs, atleast 11 PBRTs, at least 12 PBRTs, at least 13 PBRTs, at least 14 PBRTs,at least 15 PBRTs, at least 16 PBRTs, at least 17 PBRTs, at least 18PBRTs, at least 19 PBRTs, at least 20 PBRTs, or 20 or more PBRTs, all ofwhich are located N-terminally to a CS. In certain embodiments, a PBRCwill comprise 2, at least 3 PBRTs, at least 4 PBRTs, at least 5 PBRTs,at least 6 PBRTs, at least 7 PBRTs, at least 8 PBRTs, at least 9 PBRTs,at least 10 PBRTs, at least 11 PBRTs, at least 12 PBRTs, at least 13PBRTs, at least 14 PBRTs, at least 15 PBRTs, at least 16 PBRTs, at least17 PBRTs, at least 18 PBRTs, at least 19 PBRTs, at least 20 PBRTs, or 20or more PBRTs, all of which are located C-terminally to a CS.

In one embodiment, a PBRC is an amino acid sequence comprising at leastfive tandem PBRTs situated N-terminally to a capping sequence.

In one embodiment, a PBRC is an amino acid sequence comprising at leastsix to about 16 tandem PBRTs situated N-terminally to a cappingsequence.

In one embodiment, a precipitable beta roll cassette is an amino acidsequence comprising 17 or more tandem PBRTs situated N-terminally to acapping sequence.

In one embodiment, the capping sequence comprises the sequenceINAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGI EKLVEAMAQYPD(SEQ ID NO: 3) which is a C-terminal sequence on the block V beta rolldomain of the adenylate cyclase toxin of B. pertussis.

In another embodiment, a PBRC comprises the amino acid sequenceGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYINAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGIEKLVEAMAQ YPD (SEQ ID NO:4). In another embodiment, a PBRC comprises the amino acid sequenceGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYINAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGIEKLVEAM AQYPD (SEQ IDNO: 5). In another embodiment, the capping sequence comprises thesequence INAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEAIHAANQAIDPAGIEKLVEAMAQYPD (SEQ ID NO: 6) (adenylate cyclase-hemolysin [Bordetellabronchiseptica]). In another embodiment, the capping sequence comprisesthe sequence INAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEAIHAANQAIDPAGIEKLVEAMAQYPD (SEQ ID NO: 7) (adenylate cyclase-hemolysin [Bordetellabronchiseptica]). In another embodiment, the capping sequence comprisesthe sequence INAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEAIHAANQTVDPAGIEKLVEAMAQYPD (SEQ ID NO: 8) (adenylate cyclase hemolysin [Bordetellabronchiseptica]). In another embodiment, the capping sequence comprisesthe sequence QLWFSKSGSDLEVRVVGTDDAVTVAGWYSGAEHHMDSIETADGTVLLDSMVDRLVQAMAGF (SEQ ID NO: 9) (Azospirillum sp. B510 calcium binding hemolysinprotein). In another embodiment, the capping sequence comprises thesequence ADQLWFRHVGNDLEISILGTGDTATVRDWYLGSRYQIEQIRVDDGRTLVNADVEKL VQAMA(SEQ ID NO: 10) (hemolysin-type calcium-binding region Burkholderiacenocepacia MCO-3). In another embodiment, the capping sequencecomprises the sequenceADQLWFRHVGNDLEISILGSSDTATVRDWYSGSRYQIEQIRLDDGRTLVNADVEKLV QAMA (SEQ IDNO: 11) (hemolysin-type calcium-binding region [Burkholderia ambifariaMC40-6]). In another embodiment, the capping sequence comprises thesequence DARQTNLWFSQVGKDLQIDVLGSTDQVTVKDWYAGADNRVERIKTADGKTLYDSDVDKLVQAMASF (SEQ ID NO: 12) (calcium binding secreted hemolysin protein)[Herbaspirillum seropedicae SmR1]). In another embodiment, the cappingsequence comprises the sequenceDARQTNLWFSQVGKDLQIDVLGSTDQVTVKDWYAGADNRVERIKTADGKTLYDSD VDKLVQAMASF (SEQID NO: 13) (calcium binding secreted hemolysin protein [Herbaspirillumseropedicae SmR1]). In another embodiment, the capping sequencecomprises the sequenceELWFSRENNDLIIKSLLSEDKVTVQNWYSHQDHKIENIRLSNEQTLVSTQVEKMVES MAGF (SEQ IDNO: 14) (RTX toxin protein [Actinobacillus pleuropneumoniae serovar). Inanother embodiment, the capping sequence comprises the sequenceEELWFSRDGNDLQINVIGTDNQVEISDWYSGVNYQLDKVQVGDSVLLNTQLEQLVS AMASF (SEQ IDNO: 15) (hemolysin-type calcium binding protein [Shewanellapiezotolerans WP3]). In another embodiment, the capping sequencecomprises the sequenceGLSELWFSRENNDLIIKSLLSEDKVTVQNWYSHQDHKIENIRLSNEQMLVSTQVEKM VESMAGF (SEQID NO: 16) (RTX toxin protein [Actinobacillus pleuropneumoniae serovar10). In another embodiment, the capping sequence comprises the sequenceEDLWFSRDGNNLQINIIGTDDQVEVNNWYNDTNYQLDQIQVGGSVLLNNQLEQLVS AMASF (SEQ IDNO: 17) (RTX toxin [Shewanella violacea DSS12]). In another embodiment,the capping sequence comprises the sequenceELWFSRENNDLIIKSLLSEDKVTVQNWYSHQDHKIENIRLSNEQTLVSTQVEKMVES MASF (SEQ IDNO: 18) (RTX toxin protein [Actinobacillus pleuropneumoniae serovar 6).In another embodiment, the capping sequence comprises the sequenceADNFWFVKSGNDLEIDILGTHQQVTVADWFLGGSYQLQEIKAGGLELDTQVTQLVQ AMATY (SEQ IDNO: 19) (protein BRAD06535 [Bradyrhizobium sp. ORS278]). In anotherembodiment, the capping sequence comprises the sequenceELWFSRENNDLIIKSLLSEDKVTVQNWYSHQDHKIENIRLSNEQTLVSTQVEKMVES MAGF (SEQ IDNO: 20) ([Actinobacillus pleuropneumoniae L20]). In another embodiment,the capping sequence comprises the sequenceLWFSRENNDLIIKSLLSEDKVTVQNWYSHQDHKIENIRLSNEQTLVSTQVEKMVESM AGF (SEQ IDNO: 21) (ApxIVA [Actinobacillus pleuropneumoniae]). In anotherembodiment, the capping sequence comprises the sequenceLWFRKSGNNLEVSIIGTSDKLVMSNWYAGSQYQVERFQAGDGKALQANQVQSLVQ AMASF (SEQ IDNO: 22) (hemolysin-type calcium-binding protein [Xanthomonas axonopodispv. citri str. 306]). In another embodiment, the capping sequencecomprises the sequenceELWFSRENNDLIIKSLLSEDKVTVQNWYSHQDHKIENIRLSNEQTLVSTQVEKMVES MAGF (SEQ IDNO: 23) (RTX toxin IVA [Actinobacillus pleuropneumoniae])

Without wishing to be bound to theory, in some embodiments of theinvention, the ability of polypeptide comprising one or more PBRT toundergo reversible Ca2+ precipitation, can require that the one or morePBRTs be located N-terminally or C-terminally to a stabilizingpolypeptide. It is known that when a certain stabilizing polypeptides(e.g. GFP, maltose binding protein) are attached to the C-terminus ofthe beta roll, calcium-induced folding can occur (Blenner et al.,Journal of Molecular Biology. 400 (2010), pp 244-256; Szilvay et al.,Biochemistry, 48 (2009), pp 11273-11282).

Thus, in one embodiment, a PBRC is an amino acid sequence comprising oneor more PBRTs located N-terminally or C-terminally to a stabilizingpolypeptide, wherein the stabilizing polypeptide cane be, but is notlimited to, glutathione S-transferase (GST), maltose E binding protein(MBP), Green Fluorescent Protein (GFP), and variants thereof. In still afurther embodiment, the stabilizing polypeptide is an amino acidsequence of any amino acid composition wherein the sequence comprises atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 26, at least27, at least 28, at least 29, at least 30, at least 31, at least 32, atleast 33, at least 34, at least 35, at least 36, at least 37, at least38, at least 39, at least 40, at least 41, at least 42, at least 43, atleast 44, at least 45, at least 46, at least 47, at least 48, at least49, at least 50, at least 51, at least 52, at least 53, at least 54, atleast 55, at least 56, at least 57, at least 58, at least 59, at least60, at least 61, at least 62, at least 63, at least 64, at least 65, atleast 66, at least 67, or at least 68 amino acids.

In another aspect of the invention, the PBRCs described herein canfurther comprise one or more cleavage sites. In certain embodiments, thecleavage site can be positioned C-terminally or N-terminally to a PBRCso as to allow for cleavage of a PBRC from a linked purification moiety(e.g. a polypeptide purification moiety linked to a PBRC as part of afusion protein). In embodiments where the PBRC comprises more than onecleavage site, a first cleavage site can be positioned C-terminally orN-terminally to a PBRC so as to allow for cleavage of a PBRC from alinked purification moiety (e.g. a polypeptide purification moietylinked to a PBRC as part of a fusion protein) and a second cleavage sitecan be positioned between a PBRT and a capping sequence or a PBRT and astabilizing polypeptide so as to allow so as to allow for cleavage ofthe capping sequence or the stabilizing polypeptide from the one or morePBRTs in the PBRC. In certain embodiments, cleavage at such cleavagesites can be useful for purification of a purification moiety ofinterest.

In one embodiment, the cleavage site is a proteolytic cleavage site.Exemplary proteolytic cleavage sites, include, but are not limited toFactor Xa, thrombin, or enterokinase. In another embodiment, thecleavage site is a signal peptidase cleavage site. In anotherembodiment, the cleavage site is a self cleaving intein cleavage site(Amitai et al., Proceedings of the National Academy of Sciences, vol.106, no. 27, pp. 11005-11010, July 2009; Hiraga et al., Journal ofMolecular Biology, vol. 393, no. 5, pp. 1106-1117, November 2009). Anyother specific cleavage sites known in the art can be used in connectionwith the methods described herein.

The PBRTs or PBRCs described herein may be linked to a purificationmoiety by any means known in the art. The PBRTs or PBRCs describedherein ca be located at any site in a polypeptide comprising apurification moiety of interest, including a location that isN-terminal, a location that is C-terminal or a location within thesequence of the purification moiety of interest.

In addition to fusion proteins comprising the PBRTs or PBRCs describedherein, the PBRTs or PBRCs described herein can also be chemicallylinked to purification moieties other than by means of a fusion protein.Thus, reference to a PBRC linked purification moiety, or to a PBRTlinked purification moiety encompasses for purification moieties linkedto a PBRC or PBRT by peptide linkage (e.g. as a fusion protein) or bynon-peptide bond chemical linkage.

The chemical modification of PBRTs or PBRCs described herein can beperformed according to any method known in the art. For example, amidesof the PBRTs or PBRCs described herein can be prepared by techniqueswell known in the art for converting a carboxylic acid group orprecursor, to an amide. One method for amide formation at the C-terminalcarboxyl group is to cleave the polypeptide, or fusion thereof from asolid support with an appropriate amine, or to cleave in the presence ofan alcohol, yielding an ester, followed by aminolysis with the desiredamine.

Salts of carboxyl groups of the PBRTs or PBRCs described herein can beprepared by contacting the polypeptide, or fusion thereof with one ormore equivalents of a desired base such as, for example, a metallichydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonatebase such as, for example, sodium carbonate or sodium bicarbonate; or anamine base such as, for example, triethylamine, triethanolamine, and thelike.

N-acyl derivatives of an amino group of the PBRTs or PBRCs describedherein can be prepared by utilizing an N-acyl protected amino acid forthe final condensation, or by acylating a protected or unprotectedpolypeptide, or fusion thereof. O-acyl derivatives can be prepared, forexample, by acylation of a free hydroxy polypeptide or polypeptideresin. Either acylation can be carried out using standard acylatingreagents such as acyl halides, anhydrides, acyl imidazoles, and thelike. Both N- and O-acylation can be carried out together, if desired.

Formyl-methionine, pyroglutamine and trimethyl-alanine can besubstituted at the N-terminal residue of PBRTs or PBRCs describedherein. Other amino-terminal modifications include aminooxypentanemodifications.

Such chemical linkages can be useful for purifying non-peptide moleculessuch as lipids, oligonucleotides and carbohydrates, small organic orinorganic molecules, proteins, single-stranded or double-strandedoligonucleotides, polynucleotides, metals (e.g. cobalt, zinc, nickel orcopper) and the like. The chemically modified PBRTs or PBRCs describedherein can be assayed for the ability to bind to Ca2+, undergoreversible precipitation in the presence of Ca2+, or induce reversibleprecipitation of a purification moiety linked to the PBRT or PBRC usingmethods known to those skilled in the art.

The PBRTs and PBRCs described herein can also be coupled with aradioisotope or enzymatic label to facilitate their detection. Forexample, the PBRTs or PBRCs described herein can be isotopically-labeledwhere one or more atoms are replaced or substituted by an atom having anatomic mass or mass number different from the atomic mass or mass numbertypically found in nature (i.e., naturally occurring). Suitableradionuclides that may be incorporated in compounds of the presentinvention include but are not limited to ²H (also written as D fordeuterium), ³H (also written as T for tritium), ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N,¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵Iand ¹³¹I. The radionuclide that is incorporated in the instantradio-labeled compounds can depend on the specific application of thatradio-labeled compound.

Alternatively, the PBRTs or PBRCs described herein can be enzymaticallylabeled with, for example, horseradish peroxidase, alkaline phosphatase,or luciferase, and the enzymatic label detected by determination ofconversion of an appropriate substrate to product. In anotherembodiment, the PBRTs or PBRCs described herein can be labeled with afluorescent dye, spin label, heavy metal or radio-labeled peptides.

Esters of carboxyl groups of the PBRTs or PBRCs described herein canalso be prepared by any of the usual methods known in the art.

The methods and compositions described herein are useful in a broadrange of bioseparation applications. The methods and compositionsdescribed herein can be used for rapid expression and purification of apurification moiety linked to a PBRC. Such PBRC linked purificationmoieties can be expressed in any number of expression systems, includingin vitro and in vivo expression systems. Exemplary in vivo expressionsystems suitable for expressing the PBRC linked purification moietiesdescribed herein, include, but are not limited to, bacterial systems,yeast systems, and mammalian systems.

In one aspect, the invention relates to a method for purifyingpurification moieties (e.g. a PBRC or a purification moiety linked to aPBRC). In one aspect, the methods described herein can be used forpurifying one or more purification moieties from a heterogeneous mixtureof biomaterials in a sample. In another aspect, the methods descriedherein can be used to purify chemically synthesized purificationmoieties or in-vitro synthesized purification moieties.

In certain embodiments, the bioseparation methods described herein cancomprise expressing a PBRT linked purification moiety (e.g. a PBRCfusion protein) in a cellular expression system (e.g. a bacterial cell).The PBRC linked purification moiety can then be released into a mediumby cell lysis. Any method of cell lysis known in the art can be used inconjunction with the methods described herein, including, but notlimited to chemical lysis (e.g. detergents) or physical methods (e.g.sonication or French press). In certain embodiments, the PBRC or PBRClinked purification moiety can be expressed in an in-vitro expressionsystem (e.g. a rabbit reticulocyte system) such that the purificationmoiety is expressed into the expression system medium.

After expression of the PBRC linked purification moiety, bioseparationcan be achieved by increasing the free Ca2+ concentration in the mediumcomprising the PBRC linked purification moiety to induce precipitationof the PBRC linked purification moiety, followed by removing materialthat does not precipitate from the medium and then resuspending theprecipitated material in a medium having a reduced Ca2+ concentration orin a medium having a reduced concentration of free Ca2+ (e.g. a mediumcomprising a Ca2+ chelators such as EDTA). These steps can be repeateduntil the desired level of purity is reached.

In another embodiment, the PBRC linked purification moiety can beexpressed in a cellular expression system, and bioseparation can beachieved by increasing the free Ca2+ concentration within the cell priorto cellular lysis. Many methods for increasing intracellular Ca2+concentrations are known in the art, including, but not limited toadding Ca2+ to a cellular medium, with or without presence of ionophoresor cell permeabilization agents. In such embodiments, the cells can thenbe subjected to lysis conditions (e.g. chemical lysis or physical lysis)and the resulting precipitate can be recovered. Precipitated PBRC or thePBRC linked purification moieties can then be recovered by reducing thefree Ca2+ concentration (e.g. through the addition of a Ca2+ chelator)to induce solubilization of the purification moieties from theprecipitate and bioseparation can be achieved by eliminating theprecipitate. Precipitation and solubilization steps can be repeateduntil a desired level of purity is reached.

In still other embodiments, the PBRC linked purification moietiesdescribed herein can further comprise a peptide sequence to inducesecretion into the periplasm of a cell (e.g. an E. coli cell) or to themedium outside of a cell. Where the PBRC linked purification moiety issecreted to the periplasm of a cell, cell lysis may be required forfurther purification of the PBRC linked purification moiety. Where thePBRC linked purification moiety is secreted into the medium outside ofthe cell, purification can be achieved without cell lysis by eliminatingintact cells (e.g. by centrifugation) and purification of the PBRClinked purification moiety from the extracellular medium by increasingthe free Ca2+ concentration of the supernatant.

The adjustment of conditions during the purification process can beachieved by numerous methods, including, but not limited to, adjustingthe temperature, pH or salt concentration of the aqueous media.

The methods described herein can be used to purify purification moietiesof any size. A purified PBRT linked purification moiety can contain lessthan about 50%, less than about 75%, or less than about 90%, of thematerials with which it was originally associated.

In one embodiment, a purification moiety of interest can be linked toPBRC comprising a cleavable peptide sequence (e.g. a self-cleavingpeptide sequence such an intein) positioned between the PBRC and thepurification moiety. Once the PBRC linked purification moiety isexpressed in an expression system, the PBRC linked purification moietycan be recovered using standard techniques as either a homogenousmixture or as a heterogeneous sample. The mixture can then be exposed tocalcium to induce precipitation of the PBRC linked purification moiety.The PBRC linked purification moiety can then be resuspended in bufferthat has reduced Ca2+, that has reduced free Ca2+ or that contains acalcium chelator (e.g. EDTA). The PBRC linked purification moiety canthen be subjected condition that cause cleavage to separate the PBRCfrom the purification moiety and calcium can be once again added to themixture. This will precipitate out the PBRC moiety and thereby leavingbehind a sample of purified purification moiety of interest.

In another embodiment, PBRC linked purification moiety can be a moietywhich binds a second molecule and the second molecule can be used toremove the purification moiety from the sample (e.g. a resin or beadscoated with the second molecule) after induced cleavage at a sitebetween the purification moiety and the PBRC.

In some embodiments, immobilization of the PBRC linked purificationmoieties described herein or its binding proteins can be used tofacilitate separation of complexes from uncomplexed forms of one or bothof the proteins, as well as to accommodate automation of the assayImmobilization of the PBRC linked purification moieties described hereincan be by linking to a solid support, including a plastic or glass plateor bead, a chromatographic resin, a filter or a membrane. Methods ofattachment of proteins, or membranes containing same, to such supportsare well known in the art Immobilization of the PBRC linked purificationmoieties described herein can also be accomplished in any vesselsuitable for containing the reactants. Examples include microtiterplates, test tubes, and micro-centrifuge tubes.

In one embodiment, a fusion protein can be provided which adds a domainthat allows the PBRT linked purification moiety described herein to bebound to a matrix. For example, glutathione-S-transferase fusionproteins can be adsorbed onto glutathione sepharose beads or glutathionederivatized microtiter plates, which are then combined with the celllysates, and the mixture incubated under conditions conducive to complexformation. Following incubation, the beads can be washed to remove anyunbound fraction. Alternatively, the complexes can be dissociated fromthe matrix using standard electrophoretic techniques.

The methods described herein depend, in part on the finding that PBRCsundergo a reversible Ca2+ binding dependent transition. PBRCs or PBRClinked purification moieties undergo reversible precipitation at a Ca2+concentration (or free Ca2+) phase transition concentration.

The transition concentrations reversible and the isolated precipitablebeta-roll tags or purification moieties comprising a precipitablebeta-roll tag can be completely resolubilized in a medium below acertain Ca2+ concentration (or free Ca2+) transition concentration,through, for example the addition of a calcium chelator into the mediumcomprising the PBRC or PBRC linked purification moiety.

The concentration of Ca2+ required to induce reversible precipitation ofa PBRC or PBRC linked purification moiety can be readily determined byadding increasing amounts of Ca2+ until such time as the PBRC or PBRClinked purification moiety begins to precipitate from a the medium. Onecan readily determine the extent of precipitation by centrifuging themedium. In one embodiment, the amount of Ca2+ required to inducereversible precipitation a PBRC of PBRC linked purification moiety willbe about 1 mM Ca2+, more than about 1 mM Ca2+, more than about 5 mMCa2+, more than about 10 mM Ca2+, more than about 20 mM Ca2+, more thanabout 30 mM Ca2+, more than about 50 mM Ca2+, more than about 75 mMCa2+, more than about 100 mM Ca2+, more than about 150 mM Ca2+, morethan about 200 mM Ca2+, or more than about 500 mM Ca2+. In certainembodiments, the amount of Ca2+ required to induce precipitation of aPBRC or a PBRC linked purification moiety can increase as a function ofthe number of PBRTs in the PBRC. For example, a PBRC linked purificationmoiety comprising 8 PBRTs may precipitate in 150 mM Ca2+ wherein a PBRClinked purification moiety comprising 17 PBRTs may precipitate in 25 mMCa2+. One of skill in the art will readily be capable of determining theamount of Ca2+ required to precipitate a particular PBRC or a particularPBRC linked purification moiety simply by titrating increasingconcentrations of Ca2+.

The concentration of Ca2+ required to reverse precipitation of a PBRT orPBRC or of a PBRT or PBRC linked purification moiety can be readilydetermined by reducing the concentration of free Ca2+ in a medium untilsuch time as a precipitated PBRC or PBRC linked purification moietybegins to solubilize into the medium. One can readily determine theextent of precipitation by centrifuging the medium. In one embodiment,the amount of free Ca2+ in the medium required to solubilize aprecipitated PBRT or PBRC or of a PBRT or PBRC linked purificationmoiety will be less than about 1 mM Ca2+, less than about 1 mM Ca2+,less than about 5 mM Ca2+, less than about 10 mM Ca2+, less than about20 mM Ca2+, less than about 30 mM Ca2+, less than about 50 mM Ca2+, lessthan about 75 mM Ca2+, less than about 100 mM Ca2+, less than about 150mM Ca2+, less than about 200 mM Ca2+, or less than about 500 mM Ca2+. Incertain embodiments, the free Ca2+ concentration required to reverseprecipitation of a PBRC or a PBRC linked purification moiety cancorrelated to the number of PBRTs in the PBRC. For example, a PBRClinked purification moiety comprising 8 PBRTs may become soluble in ahigher free Ca2+ concentration as compared to a PBRC linked purificationmoiety comprising 17 PBRTs. One of skill in the art will readily becapable of determining the Ca2+ concentration required to reverseprecipitation a particular PBRC or a particular PBRC linked purificationmoiety simply by decreasing free Ca2+ concentrations.

The free Ca2+ concentration of a medium comprising a PBRC or a PBRClinked purification moiety can be reduced by adding one or more calciumchelators into the medium. Any number of calcium chelators can be usedin the connection with the methods described herein. Examples ofsuitable calcium chelators include, but are not limited to EDTA, EGTA,and BAPTA. In one embodiment, the amount of a calcium chelator requiredto solubilize a precipitated PBRT or PBRC or of a PBRT or PBRC linkedpurification moiety will be about 1 mM Ca2+, more than about 1 mM Ca2+,more than about 5 mM Ca2+, more than about 10 mM Ca2+, more than about20 mM Ca2+, more than about 30 mM Ca2+, more than about 50 mM Ca2+, morethan about 75 mM Ca2+, more than about 100 mM Ca2+, more than about 150mM Ca2+, more than about 200 mM Ca2+, or more than about 500 mM Ca2+.

In addition to temperature and ionic strength, other environmentalvariables useful for modulating the solubility of PBRT or PBRC or of thePBRT linked purification moieties described herein include pH, theaddition of organic solutes and solvents, side-chain ionization orchemical modification, and pressure.

The PBRC linked purification moieties described herein can be furtherpurified or isolated according to any method of protein purification orisolation known in the art. For example, PBRCs or PBRC linkedpurification moieties can be purified by various methods including,without limitation, preparative disc-gel electrophoresis, isoelectricfocusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange andpartition chromatography, precipitation and salting-out chromatography,extraction, and countercurrent distribution. For some purposes, thePBRCs or the PBRC linked purification moieties can be produced in arecombinant system in which the protein contains an additional sequencetag that facilitates purification, such as, but not limited to, apolyhistidine sequence, or a sequence that specifically binds to anantibody, such as FLAG and GST. In one embodiment, the PBRCs or the PBRClinked purification moieties can be purified from a crude lysate of thehost cell by chromatography on an appropriate solid-phase matrix.Alternatively, antibodies produced against the PBRTs or PBRCs, or a PBRClinked purification moiety or against polypeptides derived therefrom canbe used as purification reagents.

The methods and compositions described herein can be useful for thedetection of a broad range of purification moieties in biosensingapplications. For example, the methods and compositions described hereincan be used for the separation of protein of interest from a sample fordetection of bimolecular interactions. In one embodiment, if a PBRC islinked to an antibody, the PBRC linked antibody can be added to asample. Ca2+ can then be added to the sample to induce precipitation ofthe antibody such that antigen that interact with, or form a complexwith, the antibody also precipitate upon the addition of Ca2+. Theprecipitate can then be collected and resuspended and the sample can becharacterized. The presence and quantity of the target in the originalsample, as well as any associated purification moieties, can becharacterized and determined. Any antibodies, antibody fragments,antibody configurations, classes, or subclasses known in the art can beused in connection with the methods described herein. In anotherembodiment, if a PBRC is fused to a polypeptide capable of binding to asecond purification moiety, it can be added to a sample. Ca2+ can thenbe added to the sample to induce precipitation of the polypeptide suchthat other purification moieties that interact with, or form a complexwith, the polypeptide also precipitate upon the addition of Ca2+. Theprecipitate can then be collected and resuspended and the sample can becharacterized. The presence and quantity of the target in the originalsample, as well as any associated additional purification moieties, canbe characterized and determined.

The PBRC linked purification moieties described herein can be producedin prokaryotic or eukaryotic host cells by expression of nucleic acidsencoding a polypeptide of this invention. The production of thesepolypeptides can also be done as part of a larger polypeptide.

The PBRC linked purification moieties described herein can also besynthesized in vitro, e.g., by the solid phase polypeptide syntheticmethod or by recombinant DNA approaches described herein. The solidphase polypeptide synthetic method is an established and widely usedmethod. These PBRC or PBRC linked purification moieties described hereincan be further purified by fractionation on immunoaffinity orion-exchange columns; ethanol precipitation; reverse phase HPLC;chromatography on silica or on an anion-exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; or ligand affinitychromatography.

The PBRC linked purification moieties described herein can also beproduced using any in-vitro expression system known in the art or can besynthesized by chemical methods. Methods for expression of heterologousproteins in recombinant hosts, chemical synthesis of polypeptides, andin vitro translation are well known in the art and are described furtherin Sambrook J et al.; Berger and Kimmel, Methods in Enzymology, Volume152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc.,San Diego, Calif.; Gutte B and Merrifield R B, J. Am. Chem. Soc.91:501-02 (1969); Chaiken I M, CRC Crit. Rev. Biochem. 11:255-301(1981); Kaiser E T et al., Science 243:187-92 (1989); Merrifield B,Science 232:341-47 (1986); Kent S B H, Ann. Rev. Biochem. 57:957-89(1988); Offord, R. E. (1980) Semisynthetic Proteins, Wiley Publishing.Exemplary peptide synthesis methods known in the art include, but arenot limited to those described in Stewart et al., Solid Phase PeptideSynthesis, Pierce Biotechnology, Inc., Rockford, Ill., 1984; Bodanszky,Principles of Peptide Synthesis, Springer-Verlag, New York, 1984; andPennington et al., Peptide Synthesis Protocols, Humana Press, Totowa,N.J., 1994). Additionally, many companies offer custom peptide synthesisservices.

The PBRC linked purification moieties described herein can also beproduced by direct chemical synthesis. For example, the PBRC linkedpurification moieties described herein can be produced as modifiedpolypeptides, with nonpeptide moieties attached by covalent linkage tothe N-terminus and/or C-terminus. In certain embodiments, either thecarboxy-terminus or the amino-terminus, or both, are chemicallymodified. Common modifications of the terminal amino and carboxylgroups, include, but are not limited to acetylation and amidation,respectively. Amino-terminal modifications such as acylation (e.g.,acetylation) or alkylation (e.g., methylation) andcarboxy-terminal-modifications such as amidation, as well as otherterminal modifications, including cyclization, can be incorporated intovarious embodiments. Certain amino-terminal and/or carboxy-terminalmodifications and/or polypeptide extensions to the core sequence canprovide advantageous physical, chemical, biochemical, andpharmacological properties, such as: enhanced stability, increasedpotency and/or efficacy, resistance to serum proteases, desirablepharmacokinetic properties, and others.

The PBRC linked purification moieties can be prepared using recombinantDNA and molecular cloning techniques. Genes encoding the PBRC linkedpurification moieties may be produced in heterologous host cells,particularly in the cells of microbial hosts. Any techniques fortransfecting host cells and purifying proteins and polypeptides known inthe art can be used in connection with the methods described herein.Exemplary epitope tags suitable for use with the methods describedherein include, but are not limited to FLAG, HA, Myc and T7 epitopetags. The PBRTs, PBRCs or PBRC linked purification moieties describedherein can be synthesized chemically using standard polypeptidesynthesis techniques.

The invention also extends to the DNA expression vector comprising DNAcoding for the PBRTs or PBRCs described herein, whether or not theencoded products further comprise a linked purification moiety. Theinvention also provides the expression vector comprising sequencescoding for a PBRT or a PBRC configured to allow insertion of a DNAsequence downstream of the sequence coding for the PBRT or the PBRC soas to facilitate production of a fusion protein comprising a PBRT or aPBRC. For example, such vectors can comprise one or more cloning sitesbetween the sequence coding for the PBRT or the PBRC to enablegeneration of an in-frame translation product. Such vectors may comprisemultiple cloning sites in any of three reading frames. Methods forgenerating such expression vectors are well known in the art.

A variety of expression systems can be used to produce the PBRCs andPBRC linked purification moieties described herein. Such expressionsystems include vector based expression systems. Exemplary vector baseexpression systems suitable for use with the methods described hereininclude, but are not limited to, chromosomal, episomal and virus-derivedvectors, e.g., vectors derived from bacterial plasmids, frombacteriophage, from transposons, from insertion elements, from yeastepisomes, from viruses such as baculoviruses, retroviruses and vectorsderived from combinations thereof such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids.

The expression system vectors may contain regulatory regions thatregulate as well as engender expression. In general, any system orvector suitable to maintain, propagate or express polynucleotide orpolypeptide in a host cell may be used for expression in this regard.Expression systems and expression vectors can contain regulatorysequences that direct high level expression of foreign proteins relativeto the growth of the host cell. Regulatory sequences are well known tothose skilled in the art and examples include, but are not limited to,those which cause the expression of a gene to be turned on or off inresponse to a chemical or physical stimulus, including the presence ofregulatory elements in the vector, for example, enhancer sequences. Anyof these could be used to construct chimeric genes for production of theany of the binding peptides of the present invention. These chimericgenes could then be introduced into appropriate microorganisms viatransformation to provide high level expression of the peptides.

A number of recombinant expression vectors can be used for expression ofthe PBRCs and PBRC linked purification moieties described herein. Forexample, the PBRT linked purification moieties described herein can beexpressed in bacterial cells such as E. coli, insect cells (e.g., usingbaculovirus expression vectors), yeast cells, amphibian cells, ormammalian cells. Suitable host cells are well known to one skilled inthe art. Alternatively, the recombinant expression vector can betranscribed and translated in vitro, using, for example T7 promoterregulatory sequences and T7 polymerase.

Examples of E. coli expression vectors include pTrc (Amann E et al.,Gene 69:301-15 (1988)) and pET 11d (Studier et al., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) pp. 60-89). Target gene expression from the pTrc vector relies onhost RNA polymerase transcription from a hybrid trp-lac fusion promoter.Target gene expression from the pET 11d vector relies on transcriptionfrom a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNApolymerase (T7 gn1). This viral polymerase is supplied by host strainsBL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gn1gene under the transcriptional control of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman S, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) pp. 119-28). Another strategy is to alter thenucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (Wada K et al., Nucleic AcidsRes. 20(Suppl.):2111-18 (1992)). Such alteration of nucleic acidsequences can be carried out by standard DNA synthesis techniques.

In another approach, a nucleic acid can be expressed in mammalian cellsusing a mammalian expression vector. Examples of mammalian expressionvectors include pCDM8 (Seed B, Nature 329:840-41 (1987)) and pMT2PC(Kaufman R J et al., EMBO J. 6:187-95 (1987)). When used in mammaliancells, the expression vector's control functions can be provided byviral regulatory elements. For example, commonly used promoters arederived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus40. For other suitable expression systems for both prokaryotic andeukaryotic cells, see chapters 16 and 17 of Sambrook, J., Fritsh, E. F.,and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

A number of these methodologies can also be applied in vivo,systemically or locally, in a complex biological system such as a human.For example, increased copy number of nucleic acids PBRC or PBRC linkedpurification moieties described herein in expressible from (by DNAtransfection), can be employed.

Nucleic acid purification moieties encoding PBRT or PBRC linkedpurification moieties described herein can be administered to cells by avariety of methods known to those of skill in the art, including, butnot restricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as biodegradable polymers,hydrogels, cyclodextrins (see for example, Gonzalez et al., BioconjugateChem. 10:1068-1074, 1999; Wang et al., International PCT PublicationNos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)ac-id (PLGA)and PLCA microspheres (see for example, U.S. Pat. No. 6,447,796 and U.S.Patent Application Publication No. US 2002130430), biodegradablenanocapsules, and bioadhesive microspheres, or by proteinaceous vectors(O'Hare and Normand, International PCT Publication No. WO 00/53722).

This invention may also be of use in the pharmaceutical/biotechnologyindustry where therapeutic compounds need to be purified in largequantities. This approach provides very pure product in a very quickmanner.

Purification moieties that can be linked to the PBRTs or PBRCs describedherein can be any purification moiety, including a biologically activeprotein (e.g., a therapeutic peptide, protein or an enzyme useful inindustrial biocatalysis).

The purification moieties suitable for use with the methods describedherein can be of widely varying types, including, for example, peptides,non-peptide proteins, lipids, oligonucleotides and carbohydrates, oralternatively a ligand-binding protein or an active fragment thereofhaving binding affinity to a molecule selected from the group consistingof small organic or inorganic molecules, proteins, peptides,single-stranded or double-stranded oligonucleotides, polynucleotides,lipids, and carbohydrates.

Suitable purification moieties include, but are not limited to,molecules useful in medicine, agriculture and other scientific andindustrial fields. For example, suitable molecules include those ofinterest in medicine, agriculture or other scientific or industrialfields. Examples of suitable proteins include enzymes utilized inreplacement therapy; hormones for promoting growth in animals, or cellgrowth in cell culture; and active proteinaceous substances used invarious applications, e.g., in biotechnology or in medical diagnostics.One of skill in the art will recognize that many types of recombinantpolypeptides can be produced using the methods described herein. Thepresent invention is not limited to any specific types of recombinantpolypeptide described herein. Instead, it encompasses any and allrecombinant polypeptides.

The PBRTs or PBRCs described herein can be joined to a purificationmoiety from any source or origin and can include a polypeptide found inprokaryotes, viruses, and eukaryotes, including fungi, plants, yeasts,insects, and animals, including mammals (e.g. humans). Purificationmoieties suitable for use with the methods described herein include, butare not limited to any polypeptide sequences, known or hypothetical orunknown, which can be identified using common sequence repositories.Examples of such sequence repositories, include, but are not limited toGenBank EMBL, DDBJ and the NCBI. Other repositories can easily beidentified by searching on the internet. Polypeptides that can beproduced using the methods described herein also include polypeptideshave at least about 60%, 70%, 75%, 80%, 90%, 95%, or at least about 99%or more identity to any known or available polypeptide (e.g., atherapeutic polypeptide, a diagnostic polypeptide, an industrial enzyme,or portion thereof, and the like).

Purification moieties suitable for use with the methods described hereininclude, but are not limited to, polypeptides comprising one or morenon-natural amino acids.

Purification moieties suitable for use with the methods described hereininclude, but are not limited to, cytokines, inflammatory molecules,growth factors, their receptors, and oncogene products or portionsthereof. Examples of cytokines, inflammatory molecules, growth factors,their receptors, and oncogene products include, but are not limited toe.g., alpha-1 antitrypsin, Angiostatin, Antihemolytic factor, antibodies(including an antibody or a functional fragment or derivative thereofselected from: Fab, Fab′, F(ab)2, Fd, Fv, ScFv, diabody, tribody,tetrabody, dimer, trimer or minibody), angiogenic molecules, angiostaticmolecules, Apolipopolypeptide, Apopolypeptide, Asparaginase, Adenosinedeaminase, Atrial natriuretic factor, Atrial natriuretic polypeptide,Atrial peptides, Angiotensin family members, Bone MorphogenicPolypeptide (BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a,BMP-8b, BMP-10, BMP-15, etc.); C-X-C chemokines (e.g., T39765, NAP-2,ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG),Calcitonin, CC chemokines (e.g., Monocyte chemoattractant polypeptide-1,Monocyte chemoattractant polypeptide-2, Monocyte chemoattractantpolypeptide-3, Monocyte inflammatory polypeptide-1 alpha, Monocyteinflammatory polypeptide-1 beta, RANTES, 1309, R83915, R91733, HCC1,T58847, D31065, T64262), CD40 ligand, C-kit Ligand, Ciliary NeurotrophicFactor, Collagen, Colony stimulating factor (CSF), Complement factor 5a,Complement inhibitor, Complement receptor 1, cytokines, (e.g.,epithelial Neutrophil Activating Peptide-78, GRO alpha/MGSA, GRO beta,GRO gamma, MIP-1 alpha, MIP-1 delta, MCP-1), deoxyribonucleic acids,Epidermal Growth Factor (EGF), Erythropoietin (“EPO”, representing apreferred target for modification by the incorporation of one or morenon-natural amino acid), Exfoliating toxins A and B, Factor IX, FactorVII, Factor VIII, Factor X, Fibroblast Growth Factor (FGF), Fibrinogen,Fibronectin, G-CSF, GM-CSF, Glucocerebrosidase, Gonadotropin, growthfactors, Hedgehog polypeptides (e.g., Sonic, Indian, Desert),Hemoglobin, Hepatocyte Growth Factor (HGF), Hepatitis viruses, Hirudin,Human serum albumin, Hyalurin-CD44, Insulin, Insulin-like Growth Factor(IGF-I; IGF-II), interferons (e.g., interferon-alpha, interferon-beta,interferon-gamma, interferon-epsilon, interferon-zeta, interferon-eta,interferon-kappa, interferon-lambda, interferon-T, interferon-zeta,interferon-omega), glucagon-like peptide (GLP-1), GLP-2, GLP receptors,glucagon, other agonists of the GLP-1R, natriuretic peptides (ANP, BNP,and CNP), Fuzeon and other inhibitors of HIV fusion, Hurudin and relatedanticoagulant peptides, Prokineticins and related agonists includinganalogs of black mamba snake venom, TRAIL, RANK ligand and itsantagonists, calcitonin, amylin and other glucoregulatory peptidehormones, and Fc fragments, exendins (including exendin-4), exendinreceptors, interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, etc.), I-CAM-1/LFA-1, KeratinocyteGrowth Factor (KGF), Lactoferrin, leukemia inhibitory factor,Luciferase, Neurturin, Neutrophil inhibitory factor (NIF), oncostatin M,Osteogenic polypeptide, Parathyroid hormone, PD-ECSF, PDGF, peptidehormones (e.g., Human Growth Hormone), Oncogene products (Mos, Rel, Ras,Raf, Met, etc.), Pleiotropin, Polypeptide A, Polypeptide G, Pyrogenicexotoxins A, B, and C, Relaxin, Renin, ribonucleic acids, SCF/c-kit,Signal transcriptional activators and suppressors (p53, Tat, Fos, Myc,Jun, Myb, etc.), Soluble complement receptor 1, Soluble I-CAM 1, Solubleinterleukin receptors (IL-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14,15), soluble adhesion molecules, Soluble TNF receptor, Somatomedin,Somatostatin, Somatotropin, Streptokinase, Superantigens, i.e.,Staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE),Steroid hormone receptors (such as those for estrogen, progesterone,testosterone, aldosterone, LDL receptor ligand and corticosterone),Superoxide dismutase (SOD), Toll-like receptors (such as Flagellin),Toxic shock syndrome toxin (TSST-1), Thymosin a 1, Tissue plasminogenactivator, transforming growth factor (TGF-alpha, TGF-beta), Tumornecrosis factor beta (TNF beta), Tumor necrosis factor receptor (TNFR),Tumor necrosis factor-alpha (TNF alpha), transcriptional modulators (forexample, genes and transcriptional modular polypeptides that regulatecell growth, differentiation and/or cell regulation), VascularEndothelial Growth Factor (VEGF), virus-like particle, VLA-4NCAM-1,Urokinase, signal transduction molecules, estrogen, progesterone,testosterone, aldosterone, LDL, corticosterone.

Additional purification moieties suitable for use with the methodsdescribed herein include, but are not limited to, enzymes (e.g.,industrial enzymes) or portions thereof. Examples of enzymes include,but are not limited to amidases, amino acid racemases, acylases,dehalogenases, dioxygenases, diarylpropane peroxidases, epimerases,epoxide hydrolases, esterases, isomerases, kinases, glucose isomerases,glycosidases, glycosyl transferases, haloperoxidases, monooxygenases(e.g., p450s), lipases, lignin peroxidases, nitrile hydratases,nitrilases, proteases, phosphatases, subtilisins, transaminase, andnucleases. In certain embodiments, such enzymes comprising a PBRT orPBRC can be used as immobilized enzymes in industrial biocatalysis. Theenzymes comprising a PBRTs or a PBRC can also be added to a solution tofacilitate biocatalysis and then reisolated from the solution.

Additional purification moieties suitable for use with the methodsdescribed herein include, but are not limited to, agriculturally relatedpolypeptides such as insect resistance polypeptides (e.g., Crypolypeptides), starch and lipid production enzymes, plant and insecttoxins, toxin-resistance polypeptides, Mycotoxin detoxificationpolypeptides, plant growth enzymes (e.g., Ribulose 1,5-BisphosphateCarboxylase/Oxygenase), lipoxygenase, and Phosphoenolpyruvatecarboxylase.

Additional purification moieties suitable for use with the methodsdescribed herein include, but are not limited to, antibodies,immunoglobulin domains of antibodies and their fragments. Examples ofantibodies include, but are not limited to antibodies, antibodyfragments, antibody derivatives, Fab fragments, Fab′ fragments, F(ab)2fragments, Fd fragments, Fv fragments, single-chain Fv fragments (scFv),diabodies, tribodies, tetrabodies, dimers, trimers, and minibodies.

Additional purification moieties suitable for use with the methodsdescribed herein include, but are not limited to, prophylactic vaccineor therapeutic vaccine polypeptides. A prophylactic vaccine is oneadministered to subjects who are not infected with a condition againstwhich the vaccine is designed to protect. In certain embodiments, apreventive vaccine will prevent a virus from establishing an infectionin a vaccinated subject. However, even if it does not provide completeprotective immunity, a prophylactic vaccine may still confer someprotection to a subject. For example, a prophylactic vaccine maydecrease the symptoms, severity, and/or duration of the disease. Atherapeutic vaccine, is administered to reduce the impact of a viralinfection in subjects already infected with that virus. A therapeuticvaccine may decrease the symptoms, severity, and/or duration of thedisease. Vaccine polypeptides include polypeptides, or polypeptidefragments from infectious fungi (e.g., Aspergillus, Candida species)bacteria (e.g. E. coli, Staphylococci aureus)), or Streptococci (e.g.,pneumoniae); protozoa such as sporozoa (e.g., Plasmodia), rhizopods(e.g., Entamoeba) and flagellates (Trypanosoma, Leishmania, Trichomonas,Giardia, etc.); viruses such as (+) RNA viruses (examples includePoxviruses e.g., vaccinia; Picornaviruses, e.g., polio; Togaviruses,e.g., rubella; Flaviviruses, e.g., HCV; and Coronaviruses), (−) RNAviruses (e.g., Rhabdoviruses, e.g., VSV; Paramyxovimses, e.g., RSV;Orthomyxovimses, e.g., influenza; Bunyaviruses; and Arenaviruses), dsDNAviruses (Reoviruses, for example), RNA to DNA viruses, i.e.,Retroviruses, e.g., HIV and HTLV, and certain DNA to RNA viruses such asHepatitis B.

Additional purification moieties suitable for use with the methodsdescribed herein include, but are not limited to, molecules thatcomprise a chemical moiety selected from the group consisting of:cytotoxins, pharmaceutical drugs, dyes or fluorescent labels, anucleophilic or electrophilic group, a ketone or aldehyde, azide oralkyne compounds, photocaged groups, tags, a peptide, a polypeptide, apolypeptide, an oligosaccharide, polyethylene glycol with any molecularweight and in any geometry, polyvinyl alcohol, metals, metal complexes,polyamines, imidizoles, carbohydrates, lipids, biopolymers, particles,solid supports, a polymer, a targeting agent, an affinity group, anyagent to which a complementary reactive chemical group can be attached,biophysical or biochemical probes, isotypically-labeled probes,spin-label amino acids, fluorophores, aryl iodides and bromides.

Reference is also made to a “variant PBRT.” A variant PBRT is a PBRTcomprising one or more amino acid substitutions any position in thesequence of SEQ ID NO: 1 wherein the substitution replaces any aminoacid in position 1 through 9 with an amino acid having a similar sidechain group, an amino acid having a similar side chain configuration, anamino acid having an evolutionary positive relatedness, or an amino acidhaving an evolutionary neutral relatedness.

As used herein, the term “variant precipitable-beta roll cassette”(PBRC) refers to an amino acid sequence comprising at least one variantPBRT. In certain embodiments, a variant PBRC will comprise at least twovariant PBRTs. In certain embodiments, a variant PBRC will comprise atleast 3 variant PBRTs, at least 4 variant PBRTs, at least 5 variantPBRTs, at least 6 variant PBRTs, at least 7 variant PBRTs, at least 8variant PBRTs, at least 9 variant PBRTs, at least 10 variant PBRTs, atleast 11 variant PBRTs, at least 12 variant PBRTs, at least 13 variantPBRTs, at least 14 variant PBRTs, at least 15 variant PBRTs, at least 16variant PBRTs, at least 17 variant PBRTs, at least 18 variant PBRTs, atleast 19 variant PBRTs, at least 20 variant PBRTs, or 20 or more variantPBRTs. In certain embodiments, the PBRCs described herein will comprisea plurality of variant precipitable beta roll tags arranged in a tandemrepeat. For example, in certain embodiments, the variant PBRCs describedherein can comprise at least 2 variant PBRTs, at least 3 variant PBRTs,at least 4 variant PBRTs, at least 5 variant PBRTs, at least 6 variantPBRTs, at least 7 variant PBRTs, at least 8 variant PBRTs, at least 9variant PBRTs, at least 10 variant PBRTs, at least 11 variant PBRTs, atleast 12 variant PBRTs, at least 13 variant PBRTs, at least 14 variantPBRTs, at least 15 variant PBRTs, at least 16 variant PBRTs, at least 17variant PBRTs, at least 18 variant PBRTs, at least 19 variant PBRTs, atleast 20 variant PBRTs, or 20 or more variant PBRTs in tandem repeat. Incertain embodiments, a PBRC can comprise at least two PBRCs separated bya linking amino acid sequence. Where a linking amino acid sequence inpresent between two PBRTs, a PBRTs located at either end of the linkingsequence can be an individual PBRT or it can be a PBRTs that is part ofa tandem arrangement.

Thus in certain embodiments, a variant PBRC will comprise at least 2variant PBRCs, at least variant 3 PBRTs, at least 4 variant PBRTs, atleast 5 variant PBRTs, at least 6 variant PBRTs, at least 7 variantPBRTs, at least 8 variant PBRTs, at least 9 variant PBRTs, at least 10variant PBRTs, at least 11 variant PBRTs, at least 12 variant PBRTs, atleast 13 variant PBRTs, at least 14 variant PBRTs, at least 15 variantPBRTs, at least 16 variant PBRTs, at least 17 variant PBRTs, at least 18variant PBRTs, at least 19 variant PBRTs, at least 20 variant PBRTs, or20 or more variant PBRTs, all of which are located N-terminally to a CS.In certain embodiments, a variant PBRC will comprise at least 2 variantPBRCs, at least variant 3 PBRTs, at least 4 variant PBRTs, at least 5variant PBRTs, at least 6 variant PBRTs, at least 7 variant PBRTs, atleast 8 variant PBRTs, at least 9 variant PBRTs, at least 10 variantPBRTs, at least 11 variant PBRTs, at least 12 variant PBRTs, at least 13variant PBRTs, at least 14 variant PBRTs, at least 15 variant PBRTs, atleast 16 variant PBRTs, at least 17 variant PBRTs, at least 18 variantPBRTs, at least 19 variant PBRTs, at least 20 variant PBRTs, or 20 ormore variant PBRTs, all of which are located C-terminally to a CS.

In certain aspects, the invention relates to a variant PBRT thatcontains one or more amino acid insertions, deletions or substitutionsas compared to the sequence of SEQ ID NO: 1 and wherein the variant PBRTretains an ability to bind to Ca2+, undergo reversible precipitation inthe presence of Ca2+, or induce reversible precipitation of apurification moiety linked to the PBRT.

Changes can be introduced by mutation into nucleic acid sequences,thereby leading to changes in the amino acid sequence of the encodedprotein, without altering the functional activity of a PBRT or a PBRC.For example, nucleotide substitutions leading to amino acidsubstitutions at non-essential amino acid residues can be made in thesequence of a PBRT or a PBRC. A non-essential amino acid residue is aresidue that can be altered from the sequence of an amino acid of thisinvention without altering the ability of the PBRT or PBRC to bind tobind to Ca2+, undergo reversible precipitation in the presence of Ca2+,or induce reversible precipitation of a purification moiety linked tothe PBRT or PBRC.

Exemplary residues which are non-essential and therefore amenable tosubstitution to generate the variant PBRTs and PBRCs described hereincan be identified by one of ordinary skill in the art by performing anamino acid alignment of two more PBRTs or PBRCs and determining residuesthat are not required for the PBRT or PBRC to bind to Ca2+, undergoreversible precipitation in the presence of Ca2+, or induce reversibleprecipitation of a purification moiety linked to the PBRT or PBRC.

Mutations can be introduced randomly along all or part of a nucleic acidsequence encoding a PBRT or PBRC, such as by saturation mutagenesis, andthe resultant mutants can be screened, for example, for their abilitybind to Ca2+, undergo reversible precipitation in the presence of Ca2+,or induce reversible precipitation of a purification moiety linked tothe variant PBRT or variant PBRC. Following mutagenesis, thepurification moiety linked to the variant PBRT or variant PBRC can beexpressed recombinantly in a host cell and the functional activity ofthe precipitable beta-roll tag can be determined using assays availablein the art for assessing binding to Ca2+, undergoing reversibleprecipitation in the presence of Ca2+, or inducing reversibleprecipitation of purification moiety linked to the variant PBRT orvariant PBRC. In certain embodiments, the variant PBRTs described hereincan comprise one or more amino acid substitutions, insertions ordeletions, wherein the variant PBRT is functionally equivalent a PBRThaving the sequence GGAGNDTLY (SEQ ID No. 1). In one embodiment, thevariant PBRT has an identical ability bind to Ca2+, undergo reversibleprecipitation in the presence of Ca2+, or induce reversibleprecipitation of a purification moiety in a manner similar to, but notnecessarily identical to a PBRC comprising only PBRTs of the sequenceGGAGNDTLY (SEQ ID NO: 1). In one embodiment, the variant PBRT has areduced ability bind to Ca2+, undergo reversible precipitation in thepresence of Ca2+, or induce reversible precipitation of a purificationmoiety in a manner similar to, but not necessarily identical to a PBRCcomprising only PBRTs of the sequence GGAGNDTLY (SEQ ID NO: 1). In oneembodiment, the variant PBRT has an increased ability bind to Ca2+,undergo reversible precipitation in the presence of Ca2+, or inducereversible precipitation of a purification moiety in a manner similarto, but not necessarily identical to a PBRC comprising only PBRTs of thesequence GGAGNDTLY (SEQ ID NO: 1).

The variant PBRCs described herein can also comprise on or more PBRTs inaddition to one or more variant PBRTs. The variant PBRCs describedherein can also be employed in any embodiments or configurationdescribed herein for a PBRC. Thus, the description of a compositioncomprising a PBRC, or a method comprising a PBRC applies equally to avariant PBRT or a variant PBRC so long as the variant PBRT or thevariant PBRC can bind to Ca2+, undergo reversible precipitation in thepresence of Ca2+, or induce reversible precipitation of a purificationmoiety in a manner similar to, but not necessarily identical to a PBRCcomprising only PBRTs of the sequence GGAGNDTLY (SEQ ID NO: 1).

In one embodiment, a variant PBRT comprises the sequence GGXGXDXXX (SEQID NO: 2) wherein X can be selected from the group consisting of:alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine andvaline. In one embodiment, the variant PBRT has a sequence of GGXGXDXXX(SEQ ID NO: 2), wherein X is not proline. In one embodiment, the variantPBRT comprises the sequence GGXGXDXXX (SEQ ID NO: 2) wherein X is anatural or non-natural amino acid comprising a modification.

In one embodiment, a variant PBRT or PBRC comprises an amino acidsequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%identity with an amino acid sequence of SEQ ID NO: 1.

As used herein, “sequence identity” means the percentage of identicalnucleotide or amino acid residues at corresponding positions in two ormore sequences when the sequences are aligned to maximize sequencematching, i.e., taking into account gaps and insertions. The comparisonof sequences and determination of percent identity between two sequencescan be accomplished using a mathematical algorithm. Techniques fordetermining sequence identity are well known to one skilled in the art,and include, for example, analysis with a sequence comparison algorithmor FASTA version 3.0t78 using default parameters (Pearson and Lipman,Proc Natl Acad Sci USA. 1988 April; 85(8):2444-8). In anothernon-limiting example, scoring of amino acid can be calculated using thePAM250 matrix as described in Dayhoff et al., (1978) in Atlas of ProteinSequence and Structure, ed. Dayhoff, M. (Natl. Biomed. Res. Found.,Silver Spring, Md.), Vol. 5, Suppl. 3, pp. 345-352.

Percent identity or percent similarity of a DNA or peptide sequence canbe determined, for example, by comparing sequence information using theGAP computer program. The GAP program utilizes the alignment method ofNeedleman et al., 1970, as revised by Smith et al., 1981. Briefly, theGAP program defines similarity as the number of aligned symbols (i.e.,nucleotides or amino acids) that are similar, divided by the totalnumber of symbols in the shorter of the two sequences. The preferredparameters for the GAP program are the default parameters, which do notimpose a penalty for end gaps. See e.g., Schwartz et al., 1979; Gribskovet al., 1986. Nucleic acids that differ due to degeneracy of the geneticcode, and still encode the PBRTs or PBRCs, described herein areencompassed by the present disclosure.

Variants can be produced by any number of methods, including but notlimited to, error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, andany combination thereof.

Variant PBRTs or variant PBRCs falling within the scope of thisinvention, can, in general, be generated by selecting substitutions thatdo not differ significantly in their effect on maintaining (a) thestructure of the polypeptide backbone in the area of the substitution,(b) the charge or hydrophobicity of the purification moiety at thetarget site, or (c) the bulk of the side chain.

In one embodiment, a variant PBRT or a variant PBRC can comprise aconservative amino acid substitution in which an amino acid residue isreplaced with an amino acid residue having a similar side chainconfiguration Amino acid residues having similar side chainconfigurations have been defined in the art within in accordance withthe following categories: basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine), aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine), aliphatic side chains (e.g.,glycine, alanine, valine, leucine, isoleucine), and sulfur-containingside chains (methionine, cysteine). Substitutions can also be madebetween acidic amino acids and their respective amides (e.g., asparagineand aspartic acid, or glutamine and glutamic acid).

In one embodiment, a variant PBRT or a variant PBRC can comprise aconservative amino acid substitution in which an amino acid residue isreplaced with an amino acid residue having a similar side chain group.Amino acid residues having similar side chain groups have been definedin the art within in accordance with the following categories: a no sidechain group (glycine), an aliphatic side chain group (alanine, valine,leucine, isoleucine, proline), a hydroxyl side chain group (serine,threonine), an acidic side chain group (aspartic acid, glutamic acid),an amide side chain group (asparagine, glutamine), a basic side chaingroup (lysine, arginine), an imidazole side chain group (histidine), anaromatic side chain group (phenylalanine, tyrosine, tryptophan), and asulfur containing side chain group (methionine, cysteine) (see Sambrooket al, (2001) Molecular Cloning: A Laboratory Manual, Volume 3, TableA7-4).

In one embodiment, a variant PBRT or a variant PBRC can comprise aconservative amino acid substitution in which an amino acid residue isreplaced an amino acid having evolutionarily positive relatedness. Aminoacids having evolutionarily positive relatedness have been defined inthe art as follows (wherein the amino acid(s) having evolutionarilypositive relatedness are indicated in parentheses): Alanine (serine,threonine, proline, glycine); Arginine (glutamine, histidine, lysine,tryptophan); Asparagine (serine, threonine, aspartic acid, glutamicacid, glutamine, histidine, lysine); Aspartic acid (threonine, glycine,asparagine, glutamine, glutamic acid, histidine); Glutamic acid(threonine, asparagine, aspartic acid, glutamine, histidine); Glutamine(asparagine, aspartic acid, glutamic acid, histidine, arginine, lysine);Glycine (serine, threonine, alanine, aspartic acid); Histidine(asparagine, aspartic acid, glutamine, arginine); Isoleucine (threonine,methionine, leucine, valine, phenylalanine); Leucine (methionine,isoleucine, valine, phenylalanine); Lysine (threonine, asparagine,glutamine, arginine); Methionine (isoleucine, leucine, valine);Phenylalanine (isoleucine, leucine, tyrosine); Proline (serine,threonine, alanine); Serine (threonine, proline, alanine, glycine,asparagine); Threonine (serine, proline, alanine, glycine, asparagine,aspartic acid, glutamic acid, lysine, isoleucine, valine); Tryptophan(arginine, tyrosine); Tyrosine (phenylalanine, tryptophan); Valine(threonine, methionine, isoleucine, leucine) (see Dayhoff et al., (1978)in Atlas of Protein Sequence and Structure, ed. Dayhoff, M., Natl.Biomed. Res. Found., Silver Spring, Md.), Vol. 5, Suppl. 3, pp.345-352).

In one embodiment, variant of a variant PBRT or a variant PBRC cancomprise a conservative amino acid substitution in which an amino acidresidue is replaced an amino acid having evolutionarily positiverelatedness. Amino acids having evolutionarily neutral relatedness havebeen defined in the art as follows (wherein the amino acid(s) havingevolutionarily neutral relatedness are indicated in parentheses):Alanine (asparagine, aspartic acid, glutamine, glutamic acid, valine);Arginine (serine, proline, asparagine, methionine); Asparagine (alanine,glycine, arginine); Aspartic acid (serine, alanine, lysine); Cysteine(serine, tyrosine); Glutamic acid (serine, alanine, glycine, lysine);Glutamine (proline, alanine); Glycine (asparagine, glutamic acid);Histidine (proline, lysine, tyrosine); Lysine (serine, asparagine,glutamic acid, histidine, methionine); Methionine (arginine, lysine,phenylalanine); Phenylalanine (methionine, tryptophan); Proline(glutamine, histidine, arginine); Serine (cysteine, aspartic acid,glutamic acid, arginine, lysine); Threonine (none); Tryptophan(phenylalanine); Tyrosine (cysteine, histidine); Valine (alanine) (seeDayhoff et al., (1978) in Atlas of Protein Sequence and Structure, ed.Dayhoff, M., Natl. Biomed. Res. Found., Silver Spring, Md.), Vol. 5,Suppl. 3, pp. 345-352).

In one embodiment of a variant PBRT, the glycine at position 1 of SEQ IDNO: 1 is not mutated.

In another embodiment of a variant PBRT, the glycine at position 1 ofSEQ ID NO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the glycine is replaced with an amino acidhaving an uncharged polar side chain configuration (e.g., asparagine,glutamine, serine, threonine, tyrosine, or cysteine).

In another embodiment of a variant PBRT, the glycine at position 1 ofSEQ ID NO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the glycine is replaced with an amino acidhaving an aliphatic side chain configuration (e.g., alanine, valine,leucine, isoleucine)

In another embodiment of a variant PBRT, the glycine at position 1 ofSEQ ID NO: 1 can be mutated to an amino acid having evolutionarilypositive relatedness such that the glycine is replaced with any ofsenile, threonine, alanine, or aspartic acid.

In another embodiment of a variant PBRT, the glycine at position 1 ofSEQ ID NO: 1 can be mutated to an amino acid having evolutionarilyneutral relatedness such that the glycine is replaced with any ofasparagine or glutamic acid.

In one embodiment of a variant PBRT, mutation of the glycine at position1 of SEQ ID NO: 1 to any of alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine wherein mutation of the glycine at position1 of SEQ ID NO: 1 will result in a precipitable beta-roll tag that iscapable of binding to Ca2+, undergo reversible precipitation in thepresence of Ca2+, or induce reversible precipitation of a purificationmoiety linked to the variant PBRT.

In one embodiment of a variant PBRT, mutation of the glycine at position1 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid whereinmutation of the glycine at position 1 of SEQ ID NO: 1 will result in aprecipitable beta-roll tag that is capable of binding to Ca2+, undergoreversible precipitation in the presence of Ca2+, or induce reversibleprecipitation of a purification moiety linked to the variant PBRT.

In one embodiment of a variant PBRT, the glycine at position 2 of SEQ IDNO: 1 can be mutated to an asparagine residue.

In one embodiment of a variant PBRT, the glycine at position 2 of SEQ IDNO: 1 can be mutated to an aspartic acid residue.

In another embodiment of a variant PBRT, the glycine at position 2 ofSEQ ID NO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the glycine is replaced with an amino acidhaving an uncharged polar side chain configuration (e.g., asparagine,glutamine, serine, threonine, tyrosine, or cysteine).

In another embodiment of a variant PBRT, the glycine at position 2 ofSEQ ID NO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the glycine is replaced with an amino acidhaving an aliphatic side chain configuration (e.g., alanine, valine,leucine, isoleucine)

In another embodiment of a variant PBRT, the glycine at position 2 ofSEQ ID NO: 1 can be mutated to an amino acid having evolutionarilypositive relatedness such that the glycine is replaced with any ofserine, threonine, alanine, or aspartic acid.

In another embodiment of a variant PBRT, the glycine at position 2 ofSEQ ID NO: 1 can be mutated to an amino acid having evolutionarilyneutral relatedness such that the glycine is replaced with any ofasparagine or glutamic acid.

In one embodiment of a variant PBRT, mutation of the glycine at position2 of SEQ ID NO: 1 to any of alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine wherein mutation of the glycine at position2 of SEQ ID NO: 1 will result in a precipitable beta-roll tag that iscapable of binding to Ca2+, undergo reversible precipitation in thepresence of Ca2+, or induce reversible precipitation of a purificationmoiety linked to the variant PBRT.

In one embodiment of a variant PBRT, mutation of the glycine at position2 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid whereinmutation of the glycine at position 2 of SEQ ID NO: 1 will result in aprecipitable beta-roll tag that is capable of binding to Ca2+, undergoreversible precipitation in the presence of Ca2+, or induce reversibleprecipitation of a purification moiety linked to the variant PBRT.

In one embodiment of a variant PBRT, the alanine at position 3 of SEQ IDNO: 1 can be mutated to a serine, glycine, or aspartic acid residue.

In one embodiment of a variant PBRT, the alanine at position 3 of SEQ IDNO: 1 can be mutated to a glutamic acid, leucine, or asparagine residue.

In one embodiment of a variant PBRT, the alanine at position 3 of SEQ IDNO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the alanine is replaced with an amino acidhaving a nonpolar side chain configuration (e.g., valine, leucine,isoleucine, proline, phenylalanine, methionine, or tryptophan).

In one embodiment of a variant PBRT, the alanine at position 3 of SEQ IDNO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the alanine is replaced with an amino acidhaving an aliphatic side chain configuration (e.g., glycine, valine,leucine, isoleucine)

In another embodiment of a variant PBRT, the alanine at position 3 ofSEQ ID NO: 1 can be mutated to an amino acid having a similar side chaingroup such that the alanine is replaced with an amino acid having analiphatic side chain group (e.g., valine, leucine, isoleucine, proline).

In another embodiment of a variant PBRT, the alanine at position 3 ofSEQ ID NO: 1 can be mutated to an amino acid having evolutionarilypositive relatedness such that the alanine is replaced with any ofserine, threonine, proline, or glycine.

In another embodiment of a variant of a precipitable beta-roll tag, theat position 3 of SEQ ID NO: 1 can be mutated to an amino acid havingevolutionarily neutral relatedness such that the alanine is replacedwith any of asparagine, aspartic acid, glutamine, glutamic acid, orvaline.

In one embodiment of a variant PBRT, mutation of the alanine at position3 of SEQ ID NO: 1 to any of glycine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine wherein mutation of the alanine at position3 of SEQ ID NO: 1 will result in a precipitable beta-roll tag that iscapable of binding to Ca2+, undergo reversible precipitation in thepresence of Ca2+, or induce reversible precipitation of a purificationmoiety linked to the variant PBRT.

In one embodiment of a variant PBRT, mutation of the alanine at position3 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid whereinmutation of the alanine at position 3 of SEQ ID NO: 1 will result in aprecipitable beta-roll tag that is capable of binding to Ca2+, undergoreversible precipitation in the presence of Ca2+, or induce reversibleprecipitation of a purification moiety linked to the variant PBRT.

In one embodiment of a variant PBRT, the glycine at position 4 of SEQ IDNO: 1 can be mutated to an alanine residue.

In another embodiment of a variant PBRT, the glycine at position 4 ofSEQ ID NO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the glycine is replaced with an amino acidhaving an uncharged polar side chain configuration (e.g., asparagine,glutamine, serine, threonine, tyrosine, or cysteine).

In another embodiment of a variant PBRT, the glycine at position 4 ofSEQ ID NO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the glycine is replaced with an amino acidhaving an aliphatic side chain configuration (e.g., alanine, valine,leucine, isoleucine)

In another embodiment of a variant PBRT, the glycine at position 4 ofSEQ ID NO: 1 can be mutated to an amino acid having evolutionarilypositive relatedness such that the glycine is replaced with any ofserine, threonine, alanine, or aspartic acid.

In another embodiment of a variant PBRT, the glycine at position 4 ofSEQ ID NO: 1 can be mutated to an amino acid having evolutionarilyneutral relatedness such that the glycine is replaced with any ofasparagine or glutamic acid.

In one embodiment of a variant PBRT, mutation of the glycine at position4 of SEQ ID NO: 1 to any of alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine wherein mutation of the glycine at position4 of SEQ ID NO: 1 will result in a precipitable beta-roll tag that iscapable of binding to Ca2+, undergo reversible precipitation in thepresence of Ca2+, or induce reversible precipitation of a purificationmoiety linked to the variant PBRT.

In one embodiment of a variant PBRT, mutation of the glycine at position4 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid whereinmutation of the glycine at position 4 of SEQ ID NO: 1 will result in aprecipitable beta-roll tag that is capable of binding to Ca2+, undergoreversible precipitation in the presence of Ca2+, or induce reversibleprecipitation of a purification moiety linked to the variant PBRT.

In one embodiment of a variant PBRT, the asparagine at position 5 of SEQID NO: 1 can be mutated to an aspartic acid or alanine residue.

In one embodiment of a variant PBRT, the asparagine at position 5 of SEQID NO: 1 can be mutated to a serine residue.

In one embodiment of a variant PBRT, the asparagine at position 5 of SEQID NO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the asparagine is replaced with an amino acidhaving an uncharged polar side chain configuration (e.g., glycine,glutamine, serine, threonine, tyrosine, cysteine)

In one embodiment of a variant PBRT, the asparagine at position 5 of SEQID NO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the asparagine is replaced with an amino acidhaving a the side chain configuration of its amide (e.g., asparticacid).

In another embodiment of a variant PBRT, the asparagine at position 5 ofSEQ ID NO: 1 can be mutated to an amino acid having a similar side chaingroup such that the asparagine is replaced with an amino acid having anamide side chain group (e.g., glutamine).

In another embodiment of a variant PBRT, the asparagine at position 5 ofSEQ ID NO: 1 can be mutated to an amino acid having evolutionarilypositive relatedness such that the asparagine is replaced with any ofserine, threonine, aspartic acid, glutamic acid, glutamine, histidine,or lysine.

In another embodiment of a variant PBRT, the asparagine at position 5 ofSEQ ID NO: 1 can be mutated to an amino acid having evolutionarilyneutral relatedness such that the asparagine is replaced with any ofalanine, glycine, or arginine.

In one embodiment of a variant PBRT, mutation of the asparagine atposition 5 of SEQ ID NO: 1 to any of glycine, alanine, arginine,aspartic acid, cysteine, glutamic acid, glutamine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, valine wherein mutation of theasparagine at position 5 of SEQ ID NO: 1 will result in a precipitablebeta-roll tag that is capable of binding to Ca2+, undergo reversibleprecipitation in the presence of Ca2+, or induce reversibleprecipitation of a purification moiety linked to the variant PBRT.

In one embodiment of a variant PBRT, mutation of the asparagine atposition 5 of SEQ ID NO: 1 is with a non-natural or synthetic amino acidwherein mutation of the asparagine at position 5 of SEQ ID NO: 1 willresult in a precipitable beta-roll tag that is capable of binding toCa2+, undergo reversible precipitation in the presence of Ca2+, orinduce reversible precipitation of a purification moiety linked to thevariant PBRT.

In one embodiment of a variant PBRT, the aspartic acid at position 6 ofSEQ ID NO: 1 can be mutated to an asparagine residue.

In one embodiment of a variant PBRT, the aspartic acid at position 6 ofSEQ ID NO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the aspartic acid is replaced with an amino acidhaving an acidic side chain configuration (e.g., glutamic acid).

In one embodiment of a variant PBRT, the aspartic acid at position 6 ofSEQ ID NO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the aspartic acid is replaced with an amino acidhaving a the side chain configuration of its amide (e.g., asparagine).

In another embodiment of a variant PBRT, the aspartic acid at position 6of SEQ ID NO: 1 can be mutated to an amino acid having a similar sidechain group such that the aspartic acid is replaced with an amino acidhaving an acidic side chain group (e.g., glutamic acid).

In another embodiment of a variant PBRT, the aspartic acid at position 6of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarilypositive relatedness such that the aspartic acid is replaced with any ofthreonine, glycine, asparagine, glutamine, glutamic acid, or histidine.

In another embodiment of a variant PBRT, the aspartic acid at position 6of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarilyneutral relatedness such that the aspartic acid is replaced with any ofserine, alanine, or lysine.

In one embodiment of a variant PBRT, mutation of the aspartic acid atposition 6 of SEQ ID NO: 1 to any of glycine, alanine, arginine,asparagine, cysteine, glutamic acid, glutamine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine wherein mutation of the aspartic acid atposition 6 of SEQ ID NO: 1 will result in a precipitable beta-roll tagthat is capable of binding to Ca2+, undergo reversible precipitation inthe presence of Ca2+, or induce reversible precipitation of apurification moiety linked to the variant PBRT.

In one embodiment of a variant PBRT, mutation of the aspartic acid atposition 6 of SEQ ID NO: 1 is with a non-natural or synthetic amino acidwherein mutation of the aspartic acid at position 6 of SEQ ID NO: 1 willresult in a precipitable beta-roll tag that is capable of binding toCa2+, undergo reversible precipitation in the presence of Ca2+, orinduce reversible precipitation of a purification moiety linked to thevariant PBRT.

In one embodiment of a variant PBRT, the threonine at position 7 of SEQID NO: 1 can be mutated to an isoleucine or valine residue.

In one embodiment of a variant PBRT, the threonine at position 7 of SEQID NO: 1 can be mutated to a leucine residue.

In one embodiment of a variant PBRT, the threonine at position 7 of SEQID NO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the threonine is replaced with an amino acidhaving an uncharged polar side chain configuration (e.g., glycine,asparagine, glutamine, serine, tyrosine, or cysteine).

In one embodiment of a variant PBRT, the threonine at position 7 of SEQID NO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the threonine is replaced with an amino acidhaving a beta-branched side chain configuration (e.g., valine,isoleucine).

In another embodiment of a variant PBRT, the threonine at position 7 ofSEQ ID NO: 1 can be mutated to an amino acid having a similar side chaingroup such that the threonine is replaced with an amino acid having anhydroxyl side chain group (e.g., serine).

In another embodiment of a variant PBRT, the threonine at position 7 ofSEQ ID NO: 1 can be mutated to an amino acid having evolutionarilypositive relatedness such that the threonine is replaced with any ofserine, proline, alanine, glycine, asparagine, aspartic acid, glutamicacid, lysine, isoleucine, or valine

In one embodiment of a variant PBRT, mutation of the threonine atposition 7 of SEQ ID NO: 1 to any of glycine, alanine, arginine,asparagine, aspartic acid, cysteine, glutamic acid, glutamine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, tryptophan, tyrosine, valine wherein mutation of thethreonine at position 7 of SEQ ID NO: 1 will result in a precipitablebeta-roll tag that is capable of binding to Ca2+, undergo reversibleprecipitation in the presence of Ca2+, or induce reversibleprecipitation of a purification moiety linked to the variant PBRT.

In one embodiment of a variant PBRT, mutation of the threonine atposition 7 of SEQ ID NO: 1 is with a non-natural or synthetic amino acidwherein mutation of the threonine at position 7 of SEQ ID NO: 1 willresult in a precipitable beta-roll tag that is capable of binding toCa2+, undergo reversible precipitation in the presence of Ca2+, orinduce reversible precipitation of a purification moiety linked to thevariant PBRT.

In one embodiment of a variant PBRT, the leucine at position 8 of SEQ IDNO: 1 can be mutated to an isoleucine residue.

In one embodiment of a variant PBRT, the leucine at position 8 of SEQ IDNO: 1 can be mutated to a phenylalanine residue.

In one embodiment of a variant PBRT, the leucine at position 8 of SEQ IDNO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the leucine is replaced with an amino acidhaving a nonpolar side chain configuration (e.g., alanine, valine,isoleucine, proline, phenylalanine, methionine, or tryptophan).

In one embodiment of a variant PBRT, the leucine at position 8 of SEQ IDNO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the leucine is replaced with an amino acidhaving an aliphatic side chain configuration (e.g., glycine, alanine,valine, or isoleucine).

In another embodiment of a variant PBRT, leucine at position 8 of SEQ IDNO: 1 can be mutated to an amino acid having a similar side chain groupsuch that the leucine is replaced with an amino acid having an aliphaticside chain group (e.g., alanine, valine, isoleucine, proline).

In another embodiment of a variant PBRT, the leucine at position 8 ofSEQ ID NO: 1 can be mutated to an amino acid having evolutionarilypositive relatedness such that the leucine is replaced with any ofmethionine, isoleucine, valine, or phenylalanine

In another embodiment of a variant PBRT, the leucine at position 8 ofSEQ ID NO: 1 can be mutated to an amino acid having evolutionarilyneutral relatedness such that the leucine is replaced with any ofserine, asparagine, glutamic acid, histidine, or methionine.

In one embodiment of a variant PBRT, mutation of the leucine at position8 of SEQ ID NO: 1 to any of glycine, alanine, arginine, asparagine,aspartic acid, cysteine, glutamic acid, glutamine, histidine,isoleucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, valine wherein mutation of the leucineat position 8 of SEQ ID NO: 1 will result in a precipitable beta-rolltag that is capable of binding to Ca2+, undergo reversible precipitationin the presence of Ca2+, or induce reversible precipitation of apurification moiety linked to the variant PBRT.

In one embodiment of a variant PBRT, mutation of the leucine at position8 of SEQ ID NO: 1 non-natural amino acid wherein mutation of leucine atposition 8 of SEQ ID NO: 1 will result in a precipitable beta-roll tagthat is capable of binding to Ca2+, undergo reversible precipitation inthe presence of Ca2+, or induce reversible precipitation of apurification moiety linked to the variant PBRT.

In one embodiment of a variant PBRT, the tyrosine at position 9 of SEQID NO: 1 can be mutated to an isoleucine or valine residue.

In one embodiment of a variant PBRT, the tyrosine at position 9 of SEQID NO: 1 can be mutated to a phenylalanine, threonine, asparagine,aspartic acid, lysine, or serine residue.

In one embodiment of a variant PBRT, the tyrosine at position 9 of SEQID NO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the tyrosine is replaced with an amino acidhaving an uncharged polar side chain configuration (e.g., glycine,asparagine, glutamine, serine, threonine, or cysteine).

In one embodiment of a variant PBRT, the tyrosine at position 9 of SEQID NO: 1 can be mutated to an amino acid having a similar side chainconfiguration such that the tyrosine is replaced with an amino acidhaving an aromatic side chain configuration (e.g., tyrosine,phenylalanine, tryptophan, or histidine).

In another embodiment of a variant PBRT, the tyrosine at position 9 ofSEQ ID NO: 1 can be mutated to an amino acid having a similar side chaingroup such that the tyrosine is replaced with an amino acid having anaromatic side chain group (e.g., phenylalanine, tryptophan).

In another embodiment of a variant PBRT, the tyrosine at position 9 ofSEQ ID NO: 1 can be mutated to an amino acid having evolutionarilypositive relatedness such that the tyrosine is replaced with any ofphenylalanine or tryptophan.

In another embodiment of a variant PBRT, the tyrosine at position 9 ofSEQ ID NO: 1 can be mutated to an amino acid having evolutionarilyneutral relatedness such that the tyrosine is replaced with any ofcysteine or histidine.

In one embodiment of a variant PBRT, mutation of the tyrosine atposition 9 of SEQ ID NO: 1 to any of glycine, alanine, arginine,asparagine, aspartic acid, cysteine, glutamic acid, glutamine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, valine whereinmutation of tyrosine at position 9 of SEQ ID NO: 1 will result in aprecipitable beta-roll tag that is capable of binding to Ca2+, undergoreversible precipitation in the presence of Ca2+, or induce reversibleprecipitation of a purification moiety linked to the variant PBRT.

In one embodiment of a variant PBRT, mutation of the tyrosine atposition 9 of SEQ ID NO: 1 is with a non-natural or synthetic amino acidwherein mutation of the tyrosine at position 9 of SEQ ID NO: 1 willresult in a precipitable beta-roll tag that is capable of binding toCa2+, undergo reversible precipitation in the presence of Ca2+, orinduce reversible precipitation of a purification moiety linked to thevariant PBRT.

As described herein, a variant PBRC can further comprise a cappingsequence. In certain embodiments, the capping sequence in a variant PBRCcan be a variant capping sequence. A variant capping sequence can be anamino acid sequence having at least 70%, at least 80%, at least 90%, atleast 95% or at least 98% identity to SEQ ID NO: 3. In anotherembodiment, a variant capping sequence is an amino acid sequence havingat least 70%, at least 80%, at least 90%, at least 95% or at least 98%identity to any of SEQ ID NO: 4-23.

In still a further embodiment, a variant capping sequence is a sequencecomprising one or more amino acid substitutions with an amino acidhaving a similar side chain group. In one embodiment, the variantcapping sequence comprises the sequence of SEQ ID NO: 3 or any of SEQ IDNO: 4-23, wherein at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 26, at least27, at least 28, at least 29, at least 30, at least 31, at least 32, atleast 33, at least 34, at least 35, at least 36, at least 37, at least38, at least 39, at least 40, at least 41, at least 42, at least 43, atleast 44, at least 45, at least 46, at least 47, at least 48, at least49, at least 50, at least 51, at least 52, at least 53, at least 54, atleast 55, at least 56, at least 57, at least 58, at least 59, at least60, at least 61, at least 62, at least 63, at least 64, at least 65, atleast 66, at least 67, or at least 68, amino acids in the sequence ofSEQ ID NO: 3 or any of SEQ ID NO: 4-23 are substituted an amino acidhaving a similar side chain group.

In still a further embodiment, a variant capping sequence is a sequencecomprising one or more amino acid substitutions with an amino acidhaving a similar side chain configuration. In one embodiment, thevariant capping sequence comprises the sequence of SEQ ID NO: 3 or anyof SEQ ID NO: 6-23, wherein at least 1, at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, at least 30, at least 31, at least32, at least 33, at least 34, at least 35, at least 36, at least 37, atleast 38, at least 39, at least 40, at least 41, at least 42, at least43, at least 44, at least 45, at least 46, at least 47, at least 48, atleast 49, at least 50, at least 51, at least 52, at least 53, at least54, at least 55, at least 56, at least 57, at least 58, at least 59, atleast 60, at least 61, at least 62, at least 63, at least 64, at least65, at least 66, at least 67, or at least 68, amino acids in thesequence of SEQ ID NO: 3 or any of SEQ ID NO: 6-23 are substituted anamino acid having a similar side chain configuration.

In still a further embodiment, a variant capping sequence is a sequencecomprising one or more amino acid substitutions with an amino acidhaving evolutionarily positive relatedness. In one embodiment, thevariant capping sequence comprises the sequence of SEQ ID NO: 3 or anyof SEQ ID NO: 6-23, wherein at least 1, at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, at least 30, at least 31, at least32, at least 33, at least 34, at least 35, at least 36, at least 37, atleast 38, at least 39, at least 40, at least 41, at least 42, at least43, at least 44, at least 45, at least 46, at least 47, at least 48, atleast 49, at least 50, at least 51, at least 52, at least 53, at least54, at least 55, at least 56, at least 57, at least 58, at least 59, atleast 60, at least 61, at least 62, at least 63, at least 64, at least65, at least 66, at least 67, or at least 68, amino acids in thesequence of SEQ ID NO: 3 or any of SEQ ID NO: 6-23 are substituted anamino acid having evolutionarily positive relatedness.

In still a further embodiment, a variant capping sequence is a sequencecomprising one or more amino acid substitutions with an amino acidhaving evolutionarily neutral relatedness. In one embodiment, thevariant capping sequence comprises the sequence of SEQ ID NO: 3 or anyof SEQ ID NO: 6-23, wherein at least 1, at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, at least 30, at least 31, at least32, at least 33, at least 34, at least 35, at least 36, at least 37, atleast 38, at least 39, at least 40, at least 41, at least 42, at least43, at least 44, at least 45, at least 46, at least 47, at least 48, atleast 49, at least 50, at least 51, at least 52, at least 53, at least54, at least 55, at least 56, at least 57, at least 58, at least 59, atleast 60, at least 61, at least 62, at least 63, at least 64, at least65, at least 66, at least 67, or at least 68, amino acids in thesequence of SEQ ID NO: 3 or any of SEQ ID NO: 4-23 are substituted anamino acid having evolutionarily neutral relatedness.

In another embodiment, the PBRT variants or PBRC variants describedherein can also comprise a non-natural amino acid. As used herein, anon-natural amino acid can be, but is not limited to, an amino acidcomprising a moiety where a chemical moiety is attached, such as analdehyde- or keto-derivatized amino acid, or a non-natural amino acidthat includes a chemical moiety. A non-natural amino acid can also be anamino acid comprising a moiety where a saccharide moiety can beattached, or an amino acid that includes a saccharide moiety. Examplesof non-classical amino acids suitable for use with the methods andcompositions described herein include, but are not limited to, D-isomersof the common amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid,gamma-Abu, epsilon-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyricacid, 3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,beta-alanine, fluoro-amino acids, designer amino acids such asbeta-methyl amino acids, C alpha-methyl amino acids, N alpha-methylamino acids, and amino acid analogs in general.

The PBRT variants or PBRC variants described herein can also compriseone or more amino acid analog substitutions, e.g., unnatural amino acidssuch as alpha alpha-disubstituted amino acids, N-alkyl amino acids,lactic acid, and the like. These analogs include phosphoserine,phosphothreonine, phosphotyrosine, hydroxyproline,gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylicacid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid,penicillamine, ornithine, citruline, alpha.-methyl-alanine,para-benzoyl-phenylalanine, phenylglycine, propargylglycine, sarcosine,.epsilon.-N,N,N-trimethyllysine, .epsilon.-N-acetyllysine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,.omega.-N-methylarginine, and other similar amino acids and imino acidsand tert-butylglycine. The ability of PBRTs or PBRCs comprising ananalog substitutions to bind to Ca2+, undergo reversible precipitationin the presence of Ca2+, or induce reversible precipitation of apurification moiety linked to the PBRT or PBRC using methods known tothose skilled in the art.

The PBRT variants or PBRC variants described herein can further comprisepolypeptide analogs, such as peptide mimetics (Fauchere J, Adv. DrugRes. 15:29 (1986); Veber D F and Freidinger R M, Trends Neurosci.8:392-96 (1985); Evans B E et al., J. Med. Chem 30:1229-39 (1987)).Generally, peptidomimetics are structurally similar to a templatepolypeptide (i.e., a polypeptide that has a biological orpharmacological activity), such as the PBRTs or PBRCs described herein,but have one or more peptide linkages replaced by a linkage selectedfrom the group consisting of: —CH.sub.2NH—, —CH.sub.2S—,—CH.sub.2-CH.sub.2-, —CH.dbd.CH— (cis and trans), —COCH.sub.2-,—CH(OH)CH.sub.2-, and —CH.sub.2SO—, by methods known in the art andfurther described in the following references: Spatola A F in “Chemistryand Biochemistry of Amino Acids, Peptides, and Proteins,” B. Weinstein,ed., Marcel Dekker, New York, p. 267 (1983); Spatola, A F, Vega Data(March 1983), Vol. 1, Issue 3, “Peptide Backbone Modifications” (generalreview); Morley J S, Trends Pharmcol. Sci. 1:463-68 (1980) (generalreview); Hudson D et al., Int. J. Pept. Prot. Res. 14:177-85 (1979)(—CH.sub.2NH—, CH.sub.2CH.sub.2-); Spatola A F et al., Life Sci.38:1243-49 (1986) (—CH.sub.2-S); Hann M M, J. Chem. Soc. Perkin Trans.1, 307-314 (1982) (—CH—CH—, cis and trans); Almquist R G et al., J. Med.Chem. 23:1392-98 (1980) (—COCH.sub.2-); Jennings-White C et al.,Tetrahedron Left. 23:2533-34 (1982) (—COCH.sub.2-); EP 0 045 665(—CH(OH)CH.sub.2-); Holladay M W et al., Tetrahedron Lett., 24:4401-04(1983) (—C(OH)CH.sub.2-); Hruby V J, Life Sci. 31:189-99 (1982)(—CH.sub.2-S—). One example of a non-peptide linkage is —CH.sub.2NH—.

Such polypeptide mimetics can have advantages over polypeptideembodiments, including, for example: more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), altered specificity (e.g., abroad-spectrum of biological activities), reduced antigenicity, andothers. Labeling of peptidomimetics can involve covalent attachment ofone or more labels, directly or through a spacer (e.g., an amide group),to non-interfering position(s) on the peptidomimetic that are predictedby quantitative structure-activity data and/or molecular modeling. Suchnon-interfering positions can be positions that do not from directcontacts with the macromolecules(s) to which the peptidomimetic binds toproduce the therapeutic effect. Derivatization (e.g., labeling) ofpeptidomimetics can be done without substantially interfering with thedesired biological or pharmacological activity of the peptidomimetic.The ability of any peptidomimetics to polypeptides can be assayed forthe ability to bind 1,4,-benzothiazepine or derivatives thereof usingmethods know to those skilled in the art.

Systematic substitution of one or more amino acids of the PBRTs or PBRCsdescribed herein with a D-amino acid of the same type (e.g., D-lysine inplace of L-lysine) can be used to generate additional PBRT and PBRTvariants.

The following methods can be used in connection with the embodiments ofthe invention.

EXAMPLES Example 1 Purification of PBRT or PBRC Linked PurificationMoieties

17 tandem repeats of the amino acid sequence GGAGNDTLY (SEQ ID NO: 1)followed by a C-terminal “capping” sequence (the sequence is providedbelow) were fused to maltose binding protein. This construct wasexpressed in E. coli and the cells are lysed, creating a complex mixtureof E. coli proteins and the Beta Roll tagged maltose binding protein.The mixture was exposed to 100 mM calcium chloride solution to form aprecipitate form. The precipitate was pelleted and resuspended incalcium-free buffer. One precipitation cycle was sufficient to generatea relatively pure protein (FIG. 1). Multiple cycles can be used toachieve better purity.

The capping sequence used in this example is:

(SEQ ID NO: 3) INAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGIEKLVEAMAQYPD

Polypeptides comprising a 5 or 17 repeat C-capped precipitable beta-rolltags were expressed as MBP fusion proteins. 50 mM calcium was added toclarified cell lysates to induce precipitation of the polypeptidescomprising a 5 or 17 repeat C-capped precipitable beta-roll tags. Theprecipitate was pelleted by centrifugation and the pellet was washedonce and resuspended in buffer with 50 mM EGTA. The lysate, thesupernatant after calcium addition, and the resuspended pellet were thenrun on an SDS-PAGE gel (FIG. 2). The results show that the methodsdescribed herein can be used to rapidly purify polypeptides comprising a5 or 17 repeat C-capped precipitable beta-roll tags. Precipitation ofpurification moieties comprising a precipitable beta-roll tag can beconfirmed by circular dichroism analysis (FIG. 3).

An intein domain can be coupled to the construct so that the cleavagereaction and subsequent second precipitation can be examined. Otherproteins, in addition to maltose binding protein can be used with thepurification protocols described herein.

Example 2 PBRT Sequence Heat Map

The heat map for the precipitable beta-roll tag sequences describedherein was determined by using BLAST to find beta roll sequences similarto the metalloprotease of S. marcescens and then quantifying thefrequency of amino acids at each of the nine positions after beta rollsequences were identified (FIG. 4). Certain positions in theprecipitable beta-roll tag are not highly variable (e.g. positions 1, 2,4, 6, and 8), whereas other positions, exhibit moderate conservation.Positions 7, 0.9 are highly variable and can be substituted with anynatural or non-natural amino acid.

Example 3 A Designed, Phase Changing PBRT-Based Peptide for EfficientBioseparations

Non-chromatographic purification techniques are of interest sincechromatography can be the most expensive step in protein purification(Przybycien et al., (2004). Alternative approaches can rely on targetedprecipitation of the protein of interest. One approach is metal chelateaffinity precipitation, where thermoresponsive copolymers can be used tospecifically precipitate out poly-histidine tagged recombinant proteins(Balan et al., 2003; Kumar et al., 2003). Another purely protein-basedapproach is the use of thermoresponsive elastin-like peptides (ELPs)that consist of tandem repeats of the sequence VPGXG (SEQ ID NO: 1365)and precipitate with small temperature increases (McPherson et al.,1996; Meyer and Chilkoti. 1999). ELPs undergo an inverse phasetransition and aggregation, which is thought to be driven by theexposure of hydrophobic patches in the peptides upon heating (Yamaoka etal., 2003). As part of a purification system, ELPs have been coupled tointein domains that have been genetically engineered into minimalself-cleaving units (Wood et al., 1999). When coupled, the ELP-inteinsystem allows for a simple two-stage purification scheme. In the firststep, precipitation of the ELP is triggered and the fusion protein ispurified. Then, the intein is induced to cleave off the target proteinand the ELP is again precipitated, leaving behind pure target protein insolution (Banki et al., 2005). While effective for many purificationapplications, the necessary heating of samples or the alternative use ofhigh salt concentrations (Fong et al., 2009) can be problematic in manysituations. Another protein-based non-chromatographic purificationscheme developed by Ding et al. relies on calcium-dependentprecipitation of an annexin B1 tag (Ding et al., 2007). As with ELPs, aself-cleaving intein is also incorporated in the fusion protein toremove the tag following purification.

The compositions and methods described herein relate to repeat scaffoldsfor protein engineering applications. Repeat scaffolds can haverepetitive secondary structures (Courtemanche and Barrick 2008; Grove etal., 2008). Some repeat scaffolds can be engineered for biomolecularrecognition, foir example, the ankyrin repeats (Binz et al., 2004). Incertain aspects, the compositions and methods described herein relate toconsensus design to improve the engineerability of such scaffolds. Whenused in connetion with the compisitions and methods described herein,consensus design can be used to identify a core repeating peptide unit(Mosavi et al., 2004; Main et al., 2003; Parmeggiani et al., 2008; Binzet al., 2003).

Described herein is a synthetic PBRT peptide, based on the naturalrepeat-in-toxin (RTX) domain that undergoes calcium-responsive,reversible precipitation. In certain embodiments, when coupled to themaltose binding protein (MBP), the calcium-responsive tag describedherein can be ued to purify a fusion protein. In certain embodimentswhere the MBP is appended to green fluorescent protein (GFP),β-lactamase, or a thermostable alcohol dehydrogenase (AdhD), theseconstructs can also be purified by calcium-induced precipitation. Incertain embodiments, protease cleavage of the precipitating tag enablesthe recovery of pure and active target protein by cycling precipitationbefore and after cleavage.

The methods and compositions described herein relate to novelstimulus-responsive repeat scaffolds for protein engineering based onthe calcium-responsive repeat-in-toxin (RTX) domain1. The RTX domain isfound in proteins secreted through the bacterial type 1 secretion system(Holland et al., 2005). The domain consists of repeats of the consensusamino acid sequence GGXGXDXUX (SEQ ID NO: 1336), where X is variable andU is a hydrophobic amino acid. One RTX domain is the block V RTX domainfrom the adenylate cyclase toxin (CyaA) of B. pertussis. The domain isintrinsically disordered in the absence of calcium and forms a β rollstructure (FIG. 13A) in the presence of calcium (Chenal et al., 2009).The block V RTX domain retains its reversible calcium-responsivenesseven when expressed separately from the larger protein (Bauche et al.,2006; Blenner et al., 2010). Previous attempts have been made to use RTXdomains in protein engineering, including incorporation into meshnetworks, design of synthetic RTX peptides, and generation ofhydrogel-forming RTX domains (Lilie et al., 2000; Ringler, P. and G. E.Schulz. 2003; Scotter et al., 2007; Dooley et al., 2012).

In certain aspects, the compositions and methods described herein relateto the design of consensus RTX domains. The frequency of amino acids ateach position of the nine amino acid repeat unit from a set ofRTX-containing proteins have examined and resulted in the identificationof a consensus PBRT sequence GGAGNDTLY (SEQ ID NO: 1) (FIG. 13B). Alibrary of consensus constructs consisting of 5, 9, 13, or 17 repeats ofthe PBRT consensus unit was created. Upon purification of a number ofthese constructs, many of them were observed to precipitate in thepresence of calcium. Therefore, it was decided to explore thepossibility of using these consensus precipitable β roll tags (PBRTs) asa tools for bioseparation.

Reported herein is the use of PBRTs to purify recombinant proteins. Amaltose binding protein (MBP)-PBRT17 fusion was first purified as aproof of principle. This MBP-PBRT17 construct was fused to greenfluorescent protein (GFP), which was used as a reporter during initialpurification experiments. β-lactamase and a thermostable alcoholdehydrogenase (AdhD) were also fused to demonstrate the feasibility ofpurifying enzymatic proteins. In certain embodiments, a specificprotease site was engineered downstream of the tag to show that targetproteins can be fully purified by protease cleavage while retainingtheir activity.

Oligonucleotides suitable for used in connection with the methods andcompositions described herein are in Table 2.

TABLE 2 Oligonucleotides Name Sequence cons_beta_15′_ggcggtgcgggcaacgataccctgtatggtggcgccgggaatgacacattatacggaggtgctggcaatgatacgctgtatggcggagcaggtaacgac_3′ (SEQ ID NO. 1345)cons_beta_25′_attcccagcaccgccataaagggtatcgttgcctgcccccccatacagcgtgtcgttaccggcgcccccatacaaagtgtcgttacctgctccgc_3′ (SEQ ID NO. 1346) cons_beta_35′_ggcggtgctgggaatgacacactgtacggcggggcgggtaacgataccctctatggtggtgctggcaatgatacactgtat_3′ (SEQ ID NO. 1346) cons1_AvaI_F5′_attaaaaactcggggatgatgatgatgacaagggcggtgcggg_3′ (SEQ ID NO. 1347)cons9_BseRI_HindIII_R5′_tttttaataagcttgaggagtattattaatacagtgtatcattgccagcac_3′(SEQID NO. 1348) cons5_BseRI_HindIII_R5′_tttttaataagcttgaggagtattattaatacagcgtgtcgttaccg_3′ (SEQ ID NO. 1349)cons1_BtsCI_F 5′_attaaaaaggatgatggcggtgcggg_3′ (SEQ ID NO. 1350)cons4_BseRI_HindIII_R5′_tttttaataagcttgaggagtattattaatacaaagtgtcgttacctgctc_3′ (SEQID NO. 1351) cons8_BseRI_HindIII_R5′_tttttaataagcttgaggagtattattaatagagggtatcgttacccgc_3′ (SEQID NO. 1352) GFP_BseRI_F5′_aatatatagaggagataataatatatgagtaaaggagaagaacttttcactgga gt_3′(SEQ ID NO. 1353) GFP_HindIII_R5′_tattataaagcttttatttgtatagttcatccatgccatgtgtaat_3′ (SEQ ID NO. 1354)blac_BseRI_F 5′_aatatatagaggagataataatatatgagtattcaacatttccgtgtcgc_3′(SEQ ID NO. 1355) blac_HindIII_R5′_tattattaagcttttattaccaatgcttaatcagtgaggcacc_3′ (SEQ ID NO. 1356)AdhD_BserI_F 5′_aaagaggaggatcatgaatatggcaaaaagggtaaatgcattcaacgacc_3′(SEQ ID NO. 1357) AdhD_HindIII_R5′_agtgccaagatttattacacacacctccttgccatctctctatcctc_3′ (SEQ ID NO. 1358)blac_entero_BseRI_F5′_aaagaggaggatcatgaatgatgatgatgacaagatgagtattcaacatttccgtgtcgcccttattc_3′ (SEQ ID NO. 1359) AdhD_entero_BseRI_F5′_aaagaggaggatcatgaatgatgatgatgacaagatggcaaaaagggtaaatgcat tcaacgacc_3′(SEQ ID NO. 1360) entero_KOI_F 5′_cctcggggatgatggtgacaagggcggtgc_3 ′(SEQ ID NO. 1361) entero_KOI_R 5′_gcaccgccatgtcaccatcatccccgagg_3′(SEQ ID NO. 1362) entero_KOII_ F 5′_ggggatgatggtgagcagggcggtgcgggc_3′(SEQ ID NO. 1363) entero_KOII_R 5′_gcccgcaccgccctgctcaccatcatcccc_3′(SEQ ID NO. 1364) Sequences are provided for all oligonucleotides usedfor cloning experiments.

Four differently sized MBP-PBRT fusions were prepared consisting of 5,9, 13, or 17 repeats of the consensus PBRT sequence (named PBRT5, PBRT9,PBRT13, and PBRT17). In order to generate the DNA fragment for PBRT9,three oligonucleotides were synthesized: cons_β_1, cons_β_2, andcons_β_3. One ng each of these oligonucleotides was mixed along with theprimers cons1_AvaIF and cons9_BseRI_HindIII_R. PCR was performed and aclean product was obtained and gel extracted. This fragment was digestedwith AvaI and HindIII and cloned into the similarly digested pMAL_c4Evector to generate pMAL_BRT9.

To generate the PBRT5 construct, pMAL_BRT9 was used as a template forPCR with the primers cons1_AvaI_F and cons5_BseRI_HindIII_R. Thisproduct was digested with AvaI and HindIII and cloned into the pMAL_c4Evector producing pMAL_BRT5.

BRT13 was produced by concatenation of four additional repeats to PBRT9.Concatenations were achieved using a recursive ligation techniquesimilar to those previously described (Meyer et al., 2002; McDaniel etal., 2010). This four repeat insert was amplified using primerscons1_BtsCI_F and cons4_BseRI_HindIII_R. The product was digested withBtsCI and HindIII and then cloned into pMAL_BRT9 cut with BseRI andHindIII to yield pMAL_BRT13. PBRT17 was produced analogously to PBRT13,except that the reverse primer cons8_BseRI_HindIII_R was used instead ofcons4_BseRI_HindIII_R.

The emGFP gene was amplified from the Invitrogen pRSET/emGFP vectorusing primers GFP_BseRI_F and GFP_HindIII_R. The β-lactamase gene wasamplified from the pMAL_c4E vector using primers βlac_BseRI_F andβlac_HindIII_R. The AdhD gene was amplified out of pWUR85 using primersAdhD_BserI_F and AdhD_HindIII_R (Campbell et al., 2010). All three ofthese inserts were digested with BseRI and HindIII and cloned intosimilarly digested pMAL_BRT17 to yield pMAL_BRT17_GFP, pMAL_BRT17_βlacand pMAL_PBRT17_AdhD.

The native enterokinase site in the pMAL_c4E vector, which sits betweenMBP and PBRT17, was knocked out in the pMAL_BRT17_βlac andpMAL_BRT17_AdhD plasmids. Two rounds of site-directed mutagenesis wererequired to change the native recognition site, DDDDK (SEQ ID NO: 1368),to DDGEQ (SEQ ID NO: 1369), which was shown to be resistant to cleavage.A novel enterokinase recognition site was also engineered downstream ofPBRT17 in these constructs to allow for purification of the untaggedprotein of interest. Full plasmid maps of all cloned constructs areavailable in FIG. 12.

E. coli cells were used for expression and cloning. One liter culturesof TB supplemented with 100 μg/mL ampicillin and 0.2% glucose wereinoculated with 10 mL of overnight culture. Cultures were grown at 37°C. with shaking at 225 RPM to an approximate OD600 of 0.5 and inducedwith 0.3 mM IPTG. Cells harboring pMAL_BRT17 and pMAL_BRT17_βlac wereallowed to express for an additional two hours and then harvested.Cultures transformed with pMAL_BRT17_GFP were transferred to a shaker at25° C. and allowed to express for an additional 16 h and then harvestedas no fluorescence was observed when expressed at 37° C. Culturestransformed with pMAL_BRT17 AdhD were allowed to express at 37° C. foran additional 16 h as previously reported (Campbell et al., 2010). Cellswere harvested after expression and resuspended in 1/20 culture volumeof 50 mM tris-HCl, pH 7.4 for precipitation purification. For amyloseresin purification, cells were resuspended in 1/20 culture volume of MBPcolumn buffer (20 mM tris-HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4). In bothcases, cells were subsequently lysed via 150 s. Lysate was thenclarified by centrifugation at 15,000 g for 30 mM at 4° C. For amyloseresin purification, clarified lysate was diluted with five volumes ofcolumn buffer and purified as previously described (Blenner et al.,2010). All subsequent steps were performed at room temperature.

For precipitation purification, clarified lysate was added to aconcentrated calcium stock according to the data presented in FIG. 14.For example, for precipitation of MBP-PBRT17 lysate in 100 mM CaCl2, 950μL of clarified lysate was added to 50 μL of 2 M CaCl2 solution. Thesample was promptly mixed by gentle pipetting, allowed to sit at roomtemperature for 2 min and then centrifuged at 16,000 g in amicrocentrifuge for 2 min. The supernatant was carefully removed and thepellet was resuspended in the same tris buffer by gentle pipetting. Theturbid solution was centrifuged and washed for four additional cycles.For the final step, the pellet was resuspended in tris buffer with aconcentration of EGTA equivalent to the original calcium concentration.Gentle pipetting was sufficient to cause the sample to redissolve asconfirmed by observation and the lack of a precipitate upon subsequentcentrifugation.

Concentrations of all purified proteins were determined by 280 nmabsorbance using extinction coefficients predicted by ExPASy(www.expasy.org). All extinction coefficients are provided inSupplementary Table 3. Recovery of MBP-PBRT17 by either amylose resinpurification or precipitation was determined solely using this method.

TABLE 3 Calculated Extinction Coefficients Construct ε, M−1 · cm−1MBP-PBRT17 91680 MBP-PBRT17-GFP 113695 MBP-PBRT₁₇-βlac 119765MBP-PBRT₁₇-AdhD 144175 AdhD 52370Calculated molar extincton coefficients are given for all proteinconstructs. The ExPASy ProtParam tool was used for calculation.

MBP-PBRT17-GFP recoveries were estimated by comparing fluorescenceemission intensity at 509 nm with excitation at 487 nm. 100-folddilutions of both clarified lysate and purified protein were made forfluorescence measurements. Purified proteins were resuspended in thesame volume as the lysate from which they were extracted, so signalswere compared directly.

For estimation of MBP-PBRT17-βlac recovery, protein was added to anitrocefin solution and the absorbance at 486 nm was trackedcorresponding to the hydrolysis of nitrocefin. 500 μL of nitrocefinsolution was prepared by placing three nitrocefin disks in 450 μL 50 mMtris-HCl, pH 7.4 and 50 μL DMSO. In each sample well, 50 μL of thissolution was mixed with 90 μL of the same tris buffer and 10 μL ofprotein sample. For each sample tested, serial dilutions from 1× to1000× were prepared from lysate and purified protein. Initial rates weredetermined by measuring the change in absorbance at 486 nm over thefirst 20% of the change in signal between the starting absorbance andthe end absorbance. The same nitrocefin stock solution was used for allsamples to account for variations in concentration.

MBP-PBRT17-AdhD recovery was also evaluated by enzymatic activity usinga protocol previously described (Campbell et al., 2010). Since this AdhDwas isolated from the hyperthermofile Pyrococcus furiosus, all sampleswere heat treated at 80° C. for 1 h prior to evaluating activity. Allassays were performed at saturated conditions of both cofactor andsubstrate, 0.5 mM NAD+ and 100 mM 2,3-butanediol, respectively. Reactionmixtures containing 2,3-butanediol and protein sample in 50 mM glycinepH 8.8 were incubated at 45° C. in a 96 well UV microplate in aspectrophotometer. Reactions were initiated by the addition of NAD+.Initial rates were calculated by following the production of NADH at 340nm. Specific activity of cleaved AdhD was calculated using an NADHextinction coefficient (ε=6.22 mM-1 cm-1).

In order to identify the consensus RTX sequence, a database of RTXcontaining proteins was constructed by searching the UniProt(www.uniprot.org) database for hemolysin-type calcium binding domains.Individual repeats were identified and the frequency of amino acids ateach of the nine repeat positions was determined (FIG. 13B). From thisresult, the repeat sequence GGAGNDTLY (SEQ ID NO: 1) was identified asthe consensus sequence. For a few of these positions, other amino acidswere found with nearly equal frequency. However, as this sequence wasfound to be effective for purification, further investigation onsequence variation was not performed. A variety of synthetic RTX domainsof different lengths (PBRT5, PBRT9, PBRT13, PBRT17) were prepared asfusions to the C terminus of MBP, with subscripts denoting the number ofrepeats. These lengths were chosen as they reflect the variability ofnaturally occurring RTX domains. Upon the addition of calcium to thepurified PBRT17 construct, there was significant precipitation out ofsolution, which was reversed upon the addition of the chelating agentEGTA.

In order to more thoroughly characterize the observed precipitationbehavior, cells were induced to express the four MBP-PBRT constructs.Clarified cell lysates were titrated with calcium to assessprecipitation behavior by mixing with CaCl2 solution at the indicatedconcentrations, followed by by centrifugation, and measurement of themass of the pellet (FIG. 14). Due to possible variations in cell growthrates and densities, all cultures were started from saturated overnightcultures and induced simultaneously. Both PBRT13 and PBRT17 precipitatedwhen calcium concentrations exceeded 25 mM. Some precipitation wasobserved from PBRT5 and PBRT9 lysate, similar to what was observed withcontrol cell lysate. Addition of an equivalent concentration of EGTAallowed the pellets to quickly dissolve again upon gentle pipetting.

While both PBRT13 and PBRT17 precipitated upon calcium addition, PBRT17formed a pellet that was easier to clarify and was therefore selectedfor further examination. Three additional constructs were prepared byfusing MBP-PBRT17 to the N terminus of GFP, β-lactamase, and AdhD (namedMBP-PBRT17-GFP and MBP-PBRT17-βlac, MBP-PBRT17-AdhD respectively). Thesethree proteins were fused to MBP to allow for amylose resinchromatography purification as a comparison technique. GFP was chosen asa reporter protein for initial purification experiments to track thelocation of the PBRT. β-lactamase and AdhD were chosen as they are wellcharacterized enzymes whose activity can be measured withstraightforward assays.

The folding of RTX domains into β rolls is highly calcium specific.Therefore, investigation was performed to determine whether theprecipitation behavior observed was also calcium-specific. To test this,MBP-PBRT17-GFP was purified on an amylose resin and diafiltered intosalt-free tris buffer Diafiltration was used as proteins are purified inhigh salt buffer for the amylose resin step and it was observed thatPBRT precipitation was significantly in high salt. This is consistentwith previous observations that RTX calcium affinity is reduced withincreasing salt concentration (Szilvay et al., 2009). Solutions ofvarious salts were added to final concentrations of 100 mM. The sampleswere then gently mixed by pipetting, allowed to sit for 2 min, andcentrifuged at 16,000 g in a microcentrifuge for 2 min. Tubes were theninverted and the presence of a pellet at the top was indicative ofprecipitation (FIG. 15). PBRT precipitation was observed to becalcium-specific, with near complete precipitation of MBP-PBRT17-GFP (asindicated by the remaining color in solution) in the presence of calciumand no precipitation with other salts.

For all 4 constructs tested, calcium concentrations greater than 25 mMwere found to cause precipitation of the fusion protein. To assess theideal calcium concentration, all 4 constructs were precipitated from 1mL of clarified cell lysate in 25, 50, 75, and 100 mM CaCl2. Pelletswere washed in salt-free tris buffer five times. Pellets were broken upupon washing, but did not redissolve until exposed to an equivalentconcentration of EGTA after the final wash. The 100 mM CaCl2 sampleswere found to not fully redissolve, so only lower CaCl2 concentrationswere tested further. A slight increase in recovery was observed at 75 mMCaCl2 (as compared with lower CaCl2 concentrations) as confirmed bySDS-PAGE. All 4 constructs were subsequently purified by precipitationwith 75 mM CaCl2 and SDS-PAGE gels were run after 5 washes (FIG. 16). Nosignificant difference was found with increasing number of washes, sofurther quantification and recovery measurements were performed onsamples washed five times. To confirm scalability, the analogousprotocol was also performed on 50 mL lysate, and comparable results wereobtained. Additionally, the reversibility of the precipitation processwas tested. It was found that addition of calcium to the redissolvedpellet in EGTA solution did yield a pellet once again. Full pellet sizewas only recovered after dialysis into EGTA-free buffer.

The recovery and functionality of the purified proteins afterprecipitation was then qualified. To assess recovery of MBP-PBRT17, thetheoretically determined extinction coefficient was used to estimateconcentration by absorbance at 280 nm (Gill and Vonhippel, 1989).Results from purifying the construct on an amylose resin were comparedwith PBRT precipitation. For MBP-PBRT17-GFP, recoveries were calculatedas the percentage of fluorescence signal of purified sample comparedwith lysate (this was normalized against control lysate). Along withtotal protein recoveries estimated by UV absorbance, recoveries of bothMBP-PBRT17-βlac and MBP-PBRT17-AdhD were estimated by comparing lysateactivity to the activity of these constructs after purification.MBP-PBRT17-βlac recoveries were calculated using activity measured bytracking the absorbance at 486 nm for the hydrolysis of nitrocefin.MBP-PBRT17-AdhD recoveries were calculated by tracking NADH formation at340 nm in saturating conditions of both substrate and cofactor. Resultsof these trials are shown in Table 4.

TABLE 4 Recovery data for three constructs tested. MBP- PBRT17MBP-PBRT17-GFP MBP-PBRT17-βlac MBP-PBRT17-AdhD Fold Fold Fold FoldCalcium, versus versus versus Activity versus Activity mM Resin ResinFluorescence Resin Recovered Resin Recovered 25 2.0 ± 0.1 2.8 ± 0.1 61 ±3% 4.1 ± 0.1 1.6 ± 0.1% 1.6 ± 0.1 3.8 ± 0.5% 50 2.3 ± 0.1 3.7 ± 0.1 86 ±6% 5.3 ± 0.2 4.0 ± 0.1% 1.7 ± 0.1 4.7 ± 0.7% 75 2.2 ± 0.2 2.8 ± 0.3 78 ±8% 5.1 ± 0.2 3.4 ± 0.1% 2.2 ± 0.1 8.3 ± 1.4% “Fold versus Resin” denotesprotein quantity recovered relative to amylose resin for equivalentloading amount. For MBP-PBRT17-GFP, MBP-PBRT17-βlac, and MBP-PBRT17-AdhDfluorescence and activity are the respective properties relative toclarified lysate. Errors represent standard deviations. All data werecollected in triplicate.

For MBP-PBRT17, calcium precipitation recovers about double the amountof protein as compared with amylose resin purification. ForMBP-PBRT17-GFP, up to 86% recovery of fluorescence was observed.MBP-PBRT17-βlac recovery from the lysate was not as high, but was still5-fold better than the amylose resin, yielding a significant quantity ofprotein. Similar results were also observed for MBP-PBRT17-AdhD,although the yields were not quite as high compared with the resin(2-fold improvement). The overall values of the activities recovered inTable 4 were all larger than the values obtained using the amylose resinpurification. It is also possible that measuring activity in crudeextracts may introduce error beyond what was accounted for in themeasurement of endogenous hydrolysis (β-lactamase) and reduction (AdhD).Table 5 lists the absolute yield of each fusion protein based on UVabsorption at 280 nm. All fusion proteins were shown to be purified inhigh yields.

TABLE 5 Absolute Protein Yields Absolute Yield (mg/L) MBP- Calcium, MBP-PBRT17- MBP- MBP-PBRT17- mM PBRT17 GFP PBRT17-βlac AdhD 25 268 ± 11 333± 12 124 ± 3 198 ± 3 50 305 ± 14 434 ± 17 160 ± 7 273 ± 9 75 295 ± 26336 ± 40 176 ± 5 214 ± 6Amount of protein recovered for each fusion construct afterprecipitation and washing. Values were determined using UV absorbance at280 nm and calculated extinction coefficients available in the Table 3.All data were collected in triplicate and errors represent standarddeviations.

In certain embodiments, the PBRTs described herein can be coupled with acleavage tag to separate the protein of interest from the PBRT. ThepMAL_c4E vector used for these assays contains a cleavable enterokinasesite between the MBP and PBRT. This recognition sequence was removed viasite-directed mutagenesis. A new enterokinase site was engineeredbetween the PBRT and the protein of interest for MBP-PBRT17-βlac andMBP-PBRT17-AdhD. Therefore, as a proof of principle, precipitationpurified MBP-PBRT17-βlac and MBP-PBRT17-AdhD was subjected to overnightcleavage by enterokinase digestion. Calcium was then added directly tothe cleavage reaction to precipitate MBP-PBRT17, thereby separating thetag from the protein of interest following centrifugation. This is shownin FIG. 17 for MBP-PBRT17-AdhD, showing pure, soluble protein bySDS-PAGE. Recoveries of 93±7% were obtained by tracking UV absorbance at280 nm, meaning 93% of the AdhD in the precipitation purified sample wasrecovered after cleavage and reprecipitation of the tag. Specificactivity of the purified enzyme was also calculated to be 20.2±1.3min-1, which is similar to what has been previously reported, indicatingthis system has little to no effect on the activities of purifiedproteins (Campbell et al., 2010). However, in the case ofMBP-PBRT17-βlac, the cleaved β-lactamase remained in the insolublefraction following enterokinase cleavage and calcium precipitation. Uponfurther investigation it was found that β-lactamase will precipitate inhigh calcium concentrations. As a control experiment, similar behaviorwas observed in recombinant β-lactamase. In 75 mM CaCl2, an insolublepellet was formed upon centrifugation. Activity assays confirmed asignificant amount of active protein in the insoluble fraction. Incertain embodiments, the protease used could be fused to theprecipitating PBRT or a self-cleaving intein could be incorporated.Fusing the protease to the PBRT can be used for its removal from thetarget protein in the final precipitation. A self-cleaving intein canalso be used to fulfill a similar function. PBRT can also precipitatewithout being fused to the MBP, indicating that the MBP is not essentialfor this system. In certain embodiments, the MBP may be useful forimproving protein expression levels.

The results described herein show a correlation between length andprecipitation (FIG. 14). However, there has not been extensive work instudying the role of the number of repeats on RTX behavior. The impactof altering the number of native. RTX repeats in the block V CyaA RTXdomain of B. pertussis was previously examined without significant sizeeffect and C-terminal capping was required for calcium-responsiveness(Shur and Banta, 2012). As for past efforts to design synthetic RTXdomains, the synthetic domains created by Scotter et al. consisted of 4RTX repeats and those prepared by Lilie et al. consisted of 8 repeats(Lilie et al., 2000; Scotter et al., 2007). The peptides create by Lilieet al. were weakly calcium-responsive, while those of Scotter et al.were only lanthanum-responsive and formed partially insoluble filamentsin the presence of lanthanum. In general, β sheets are prone toaggregation and nature uses various strategies to ensure solubility ofproteins containing these motifs (Richardson and Richardson, 2002).PBRTs may be a balance between this tendency and thecalcium-responsiveness of the β roll.

The technique described herein provides a new stimulus-responsivephase-changing peptide useful in a range of applications similar tothose for which ELPs have been used, such as recombinant proteinpurification or the creation of “smart” biomaterials. The PBRTsdescribed herein possesses certain advantages over ELPs and annexin B1since precipitation is simpler to achieve and the PBRT peptide issignificantly smaller. Additionally, PBRT17 precipitates in as little as25 mM CaCl2 at room temperature, compared to the larger ionic strengthand higher temperature increases required for ELP precipitation.Precipitation also occurs instantaneously, whereas annexin B1-basedsystems require a 2 h incubation period at 4° C. Overall, PBRTs offer anew tool for rapid purification of recombinant proteins. The protocoldescribed here can be performed to obtain purified fusion protein fromlysate in only a few minutes. Further optimization of the PBRT systemshould enable the use of specific proteases to purify target proteinsand further improve the precipitation and resolubilization process,greatly enhancing the ability to rapidly purify recombinant proteins.

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Example 4 Exemplary PBRCs

Below are the amino acid sequences of two exemplary PBRCs.

(SEQ ID NO: 4) GGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYINAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAG IEKLVEAMAQYPD(SEQ ID NO: 5) GGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYINAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAA NQAVDQAGIEKLVEAMAQYPD

Example 5 Sequences of PBRT and PBRC Peptides that do not InducePrecipitation in Response to Ca2+

The following peptides are soluble in the presence of calcium.

(SEQ ID NO: 1339) GSARDDVLIGDAGANVLNGLADNDVLSGGAGDDVLLGDEGSDLLSGDAGNDDLFGGQGDDTYLFGVGYGHDTIYESGGGHDTIRGSARDDVLIGDAGANVLNGLADNDVLSGGAGDDVLLGDEGSDLLSGDAGNDDLFGGQGDDTYLFGVGYGHDTIYESGGGHDTIRINAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGIEKLVEAMAQYPD (SEQ ID NO: 1340)GSARDDVLIGDAGANVLNGLADNDVLSGGAGDDVLLGDEGSDLLSGDAGNDDLFGGQGDDTYLFGVGYGHDTIYESGGGHDTIRGDAGANVLNGLADNDVLSGGAGDDVLLGDEGSDLLSGDAGNDDLFGGQGDDTYLFGVGYGHDTIYE SGGGHDTIR(SEQ ID NO: 1341) GSARDDVLIGDAGANVLNGLADNDVLSGGAGDDVLLGDEGSDLLSGDAGNDDLFGGQGDDTYLFGVGYGHDTIYESGGGHDTIRGDAGANVLNGLADNDVLSGGAGDDVLLGDEGSDLLSGDAGNDDLFGGQGDDTYLFGVGYGHDTIYESGGGHDTIRINAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGIEKLVEAMAQYPD (SEQ ID NO: 1341)GGSGNDVIVGNAANNVLKGGAGNDVLFGGGGADELWGGAGKDIFV (SEQ ID NO: 1342)GGSGNDVIVGNAANNVLKGGAGNDVLFGGGGADELWGGAGKDIFVFSAASDSAPGASDWIRDFQKGIDKIDLSFFNKEANSSDFIHFVDHFSGTAGEALLSYNASSNVTDLSVNIGGHQAPDFLVKIVGQVDVATDFIV

What is claimed is:
 1. A precipitable beta roll cassette (PBRC)comprising at least two beta roll tags (PBRTs) wherein the at least twoPBRTs each comprise the amino acid sequence of SEQ ID NO: 1, wherein thePBRC precipitates in response to binding of calcium by the at least twoPBRTs.
 2. A precipitable beta roll cassette (PBRC) comprising at leasttwo beta roll tags (PBRTs) wherein the at least two PBRTs areindependently any of: (a) a polypeptide having the amino acid sequenceof SEQ ID NO: 1, or (b) a polypeptide having the amino acid sequence ofany of SEQ ID NOs 25-1337; wherein the PBRC precipitates in response tobinding of calcium by the at least two PBRTs.
 3. A precipitable betaroll cassette (PBRC) comprising at least two beta roll tags (PBRTs)wherein the at least two PBRTs are independently any of: (a) apolypeptide having the amino acid sequence of SEQ ID NO: 1; or (b) apolypeptide having the amino acid sequence of any of SEQ ID NOs 25-1337;or (c) a polypeptide comprising the amino acid sequence GXXXXXXXX,wherein, (i) the X at position 2 is an amino acid selected from thegroup consisting of glycine, asparagine or aspartic acid, and (ii) the Xat position 3 is an amino acid selected from the group consisting ofalanine, glycine, aspartic acid, glutamic acid, leucine or asparagine,and (iii) the X at position 4 is an amino acid selected from the groupconsisting of glycine or alanine, and (iv) the X at position 5 is anamino acid selected from the group consisting of asparagine, asparticacid, alanine, or serine, and (v) the X at position 6 is an amino acidselected from the group consisting of aspartic acid or asparagine, (vi)the X at position 7 is an amino acid selected from the group consistingof threonine, isoleucine, valine, or leucine, and (vii) the X atposition 8 is an amino acid selected from the group consisting ofleucine, isoleucine, or phenylalanine, and (viii) the X at position 9 isan amino acid selected from the group consisting of tyrosine,isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid,lysine or serine; wherein the PBRC precipitates in response to bindingof calcium by the at least two PBRTs.
 4. The PBRC of any of claims 1-3further comprising a capping sequence.
 5. The PBRC of any of claims 1-3further comprising a stabilizing polypeptide.
 6. A PBRC linkedpurification moiety comprising the PBRC of any of claims 1-3.
 7. ThePBRC linked purification moiety of claim 6 wherein the PBRC is linked tothe purification moiety by a peptide bond.
 8. The PBRC linkedpurification moiety of claim 6 wherein the PBRC is linked to thepurification moiety by a chemical bond that is not a peptide bond. 9.The PBRC of any of claims 1-3 further comprising a cleavage site locatedN-terminally or C-terminally to one or more of the at least two PBRTs.10. The PBRC of claim 9, wherein the cleavage site is selected from thegroup comprising an intein cleavage site, a Factor Xa cleavage site, athrombin cleavage site, an enterokinase cleavage site, or a signalpeptidase cleavage site.
 11. A polypeptide comprising the PBRC of any ofclaims 1-3 and a purification moiety.